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author:("Zhou, zhikong")
1.  Pooled PCR testing strategy and prevalence estimation of submicroscopic infections using Bayesian latent class models in pregnant women receiving intermittent preventive treatment at Machinga District Hospital, Malawi, 2010 
Malaria Journal  2014;13(1):509.
Low malaria parasite densities in pregnancy are a diagnostic challenge. PCR provides high sensitivity and specificity in detecting low density of parasites, but cost and technical requirements limit its application in resources-limited settings. Pooling samples for PCR detection was explored to estimate prevalence of submicroscopic malaria infection in pregnant women at delivery. Previous work uses gold-standard based methods to calculate sensitivity and specificity of tests, creating a challenge when newer methodologies are substantially more sensitive than the gold standard. Thus prevalence was estimated using Bayesian latent class models (LCMs) in this study.
Nested PCR (nPCR) for the 18S rRNA gene subunit of Plasmodium falciparum was conducted to detect malaria infection in microscopy-negative Malawian women on IPTp. Two-step sample pooling used dried blood spot samples (DBSs) collected from placenta or periphery at delivery. Results from nPCR and histology as well as previously published data were used to construct LCMs to estimate assay sensitivity and specificity. Theoretical confidence intervals for prevalence of infection were calculated for two-step and one-step pooling strategies.
Of 617 microscopy-negative Malawian women, 39 (6.3%) were identified as actively infected by histology while 52 (8.4%) were positive by nPCR. One hundred forty (22.7%) individuals had past infection assessed by histology. With histology as a reference, 72% of women in the active infection group, 7.1% in the past infection group and 3.2% in histology-negative group were nPCR positive. Using latent class models without a gold standard, histology had a median sensitivity of 49.7% and specificity of 97.6% for active infection while PCR had a median sensitivity of 96.0% and specificity of 99.1%. The true prevalence of active infection was estimated at 8.0% (CI: 5.8-10.5%) from PCR. PCR also had similar sensitivity for detecting either peripheral or placental malaria for submicroscopic infections. One-step pooling would give similar confidence intervals for pool sizes less than 20 while reducing the number of tests performed.
Pooled nPCR testing was a sensitive and resource-efficient strategy and LCMs provided precise prevalence estimates of submicroscopic infections. Compared to two-step pooling, one-step pooling could provide similar prevalence estimates at population levels with many fewer tests required.
Electronic supplementary material
The online version of this article (doi:10.1186/1475-2875-13-509) contains supplementary material, which is available to authorized users.
PMCID: PMC4301903  PMID: 25522751
Submicroscopic malaria infection; Nested PCR; Placental histology; IPTp; Pooled sample estimates; Latent class models (LCMs)
2.  Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay 
Journal of Clinical Microbiology  2013;51(11):3666-3674.
High-throughput, sensitive, and cost-effective HIV drug resistance (HIVDR) detection assays are needed for large-scale monitoring of the emergence and transmission of HIVDR in resource-limited settings. Using suspension array technology, we have developed a multiplex allele-specific (MAS) assay that can simultaneously detect major HIVDR mutations at 20 loci. Forty-five allele-specific primers tagged with unique 24-base oligonucleotides at the 5′ end were designed to detect wild-type and mutant alleles at the 20 loci of HIV-1 subtype C. The MAS assay was first established and optimized with three plasmid templates (C-wt, C-mut1, and C-mut2) and then evaluated using 148 plasma specimens from HIV-1 subtype C-infected individuals. All the wild-type and mutant alleles were unequivocally distinguished with plasmid templates, and the limits of detection were 1.56% for K219Q and K219E, 3.13% for L76V, 6.25% for K65R, K70R, L74V, L100I, K103N, K103R, Q151M, Y181C, and I47V, and 12.5% for M41L, K101P, K101E, V106A, V106M, Y115F, M184V, Y188L, G190A, V32I, I47A, I84V, and L90M. Analyses of 148 plasma specimens revealed that the MAS assay gave 100% concordance with conventional sequencing at eight loci and >95% (range, 95.21% to 99.32%) concordance at the remaining 12 loci. The differences observed were caused mainly by 24 additional low-abundance alleles detected by the MAS assay. Ultradeep sequencing analysis confirmed 15 of the 16 low-abundance alleles. This multiplex, sensitive, and straightforward result-reporting assay represents a new efficient genotyping tool for HIVDR surveillance and monitoring.
PMCID: PMC3889749  PMID: 23985909
3.  Robust Non-Rigid Point Set Registration Using Student's-t Mixture Model 
PLoS ONE  2014;9(3):e91381.
The Student's-t mixture model, which is heavily tailed and more robust than the Gaussian mixture model, has recently received great attention on image processing. In this paper, we propose a robust non-rigid point set registration algorithm using the Student's-t mixture model. Specifically, first, we consider the alignment of two point sets as a probability density estimation problem and treat one point set as Student's-t mixture model centroids. Then, we fit the Student's-t mixture model centroids to the other point set which is treated as data. Finally, we get the closed-form solutions of registration parameters, leading to a computationally efficient registration algorithm. The proposed algorithm is especially effective for addressing the non-rigid point set registration problem when significant amounts of noise and outliers are present. Moreover, less registration parameters have to be set manually for our algorithm compared to the popular coherent points drift (CPD) algorithm. We have compared our algorithm with other state-of-the-art registration algorithms on both 2D and 3D data with noise and outliers, where our non-rigid registration algorithm showed accurate results and outperformed the other algorithms.
PMCID: PMC3950182  PMID: 24618749
4.  Temperature Sensitivity and Basal Rate of Soil Respiration and Their Determinants in Temperate Forests of North China 
PLoS ONE  2013;8(12):e81793.
The basal respiration rate at 10°C (R10) and the temperature sensitivity of soil respiration (Q10) are two premier parameters in predicting the instantaneous rate of soil respiration at a given temperature. However, the mechanisms underlying the spatial variations in R10 and Q10 are not quite clear. R10 and Q10 were calculated using an exponential function with measured soil respiration and soil temperature for 11 mixed conifer-broadleaved forest stands and nine broadleaved forest stands at a catchment scale. The mean values of R10 were 1.83 µmol CO2 m−2 s−1 and 2.01 µmol CO2 m−2 s−1, the mean values of Q10 were 3.40 and 3.79, respectively, for mixed and broadleaved forest types. Forest type did not influence the two model parameters, but determinants of R10 and Q10 varied between the two forest types. In mixed forest stands, R10 decreased greatly with the ratio of coniferous to broadleaved tree species; whereas it sharply increased with the soil temperature range and the variations in soil organic carbon (SOC), and soil total nitrogen (TN). Q10 was positively correlated with the spatial variances of herb-layer carbon stock and soil bulk density, and negatively with soil C/N ratio. In broadleaved forest stands, R10 was markedly affected by basal area and the variations in shrub carbon stock and soil phosphorus (P) content; the value of Q10 largely depended on soil pH and the variations of SOC and TN. 51% of variations in both R10 and Q10 can be accounted for jointly by five biophysical variables, of which the variation in soil bulk density played an overwhelming role in determining the amplitude of variations in soil basal respiration rates in temperate forests. Overall, it was concluded that soil respiration of temperate forests was largely dependent on soil physical properties when temperature kept quite low.
PMCID: PMC3858269  PMID: 24339966
5.  The BLOS1 interacting protein KXD1 is involved in the biogenesis of lysosome-related organelles 
Traffic (Copenhagen, Denmark)  2012;13(8):1160-1169.
Biogenesis of lysosome-related organelles complex-1 (BLOC-1) is an eight-subunit complex involved in lysosomal trafficking. Interacting proteins of these subunits expand the understanding of its biological functions. With the implementation of the naïve Bayesian analysis, we found that a human uncharacterized 20 kDa coiled-coil KxDL protein, KXD1, is a BLOS1-interacting protein. In vitro binding assays confirmed the interaction between BLOS1 and KXD1. Mouse KXD1 homolog was widely expressed and absent in Kxd1 knockout (KO) mice. BLOS1 was apparently reduced in Kxd1-KO mice. Mild defects in the melanosomes of the retinal pigment epithelia and in the platelet dense granules of the Kxd1-KO mouse were observed, mimicking a mouse model of mild Hermansky-Pudlak syndrome that affects the biogenesis of lysosome-related organelles.
PMCID: PMC3800082  PMID: 22554196
BLOC-1; KXD1; BLOS1; lysosome-related organelles; Hermansky-Pudlak syndrome
6.  Prevalence of HIV Drug Resistance Before and 1 Year After Treatment Initiation in 4 Sites in the Malawi Antiretroviral Treatment Program 
Since 2004, the Malawi antiretroviral treatment (ART) program has provided a public health–focused system based on World Health Organization clinical staging, standardized first-line ART regimens, limited laboratory monitoring, and no patient-level monitoring of human immunodeficiency virus drug resistance (HIVDR). The Malawi Ministry of Health conducts periodic evaluations of HIVDR development in prospective cohorts at sentinel clinics. We evaluated viral load suppression, HIVDR, and factors associated with HIVDR in 4 ART sites at 12–15 months after ART initiation. More than 70% of patients initiating ART had viral suppression at 12 months. HIVDR prevalence (6.1%) after 12 months of ART was low and largely associated with baseline HIVDR. Better follow-up, removal of barriers to on-time drug pickups, and adherence education for patients 16–24 years of age may further prevent HIVDR.
PMCID: PMC3338306  PMID: 22544204
7.  Differences in selective pressure on dhps and dhfr drug resistant mutations in western Kenya 
Malaria Journal  2012;11:77.
Understanding the origin and spread of mutations associated with drug resistance, especially in the context of combination therapy, will help guide strategies to halt and prevent the emergence of resistance. Unfortunately, studies have assessed these complex processes when resistance is already highly prevalent. Even further, information on the evolutionary dynamics leading to multidrug-resistant parasites is scattered and limited to areas with low or seasonal malaria transmission. This study describes the dynamics of strong selection for mutations conferring resistance against sulphadoxine-pyrimethamine (SP), a combination therapy, in western Kenya between 1992 and 1999, just before SP became first-line therapy (1999). Importantly, the study is based on longitudinal data, which allows for a comprehensive analysis that contrasts with previous cross-sectional studies carried out in other endemic regions.
This study used 236 blood samples collected between 1992 and 1999 in the Asembo Bay area of Kenya. Pyrosequencing was used to determine the alleles of dihydrofolate reductase (dhfr) and dihydropterote synthase (dhps) genes. Microsatellite alleles spanning 138 kb around dhfr and dhps, as well as, neutral markers spanning approximately 100 kb on chromosomes 2 and 3 were characterized.
By 1992, the South-Asian dhfr triple mutant was already spreading, albeit in low frequency, in this holoendemic Kenyan population, prior to the use of SP as a first-line therapy. Additionally, dhfr triple mutant alleles that originated independently from the predominant Southeast Asian lineage were present in the sample set. Likewise, dhps double mutants were already present as early as 1992. There is evidence for soft selective sweeps of two dhfr mutant alleles and the possible emergence of a selective sweep of double mutant dhps alleles between 1992 and 1997. The longitudinal structure of the dataset allowed estimation of selection pressures on various dhfr and dhps mutants relative to each other based on a theoretical model tailored to P. falciparum. The data indicate that drug selection acted differently on the resistant alleles of dhfr and dhps, as evidenced by fitness differences. Thus a combination drug therapy such as SP, by itself, does not appear to select for "multidrug"-resistant parasites in areas with high recombination rate.
The complexity of these observations emphasizes the importance of population-based studies to evaluate the effects of strong drug selection on Plasmodium falciparum populations.
PMCID: PMC3338400  PMID: 22439637
Plasmodium; Malaria; Dihydrofolate Reductase; Dihydropterote synthase; Sulphadoxine-pyrimethamine; Natural selection; Selective sweep; Drug resistance
8.  Comparison of HIV-1 resistance profiles in plasma RNA versus PBMC DNA in heavily treated patients in Honduras, a resource-limited country 
The World Health Organization currently does not recommend the use of dried blood spot specimens for drug resistance testing in patients undergoing antiretroviral therapy (ART). Therefore, HIV-1 resistance testing using peripheral blood mononuclear cells (PBMCs) may be of value in resource-limited settings. We compared genotypic resistance profiles in plasma and PBMCs from patients failing ART in two cities of Honduras (Tegucigalpa and San Pedro Sula), a resource-limited country. One hundred patients failing ART were randomly selected from a longitudinal patient monitoring cohort. Plasma and PBMC samples without patient identifier were used for genotypic resistance testing. Sequence data were analyzed, resistance profiles were determined and compared using Stanford HIV Drug Resistance Database algorithm. Specimens with concordant resistance profiles between the two compartments were 88% (95% CI: 80.3% - 94.5 %). Nine specimens (12%, 95% CI: 6.5% - 21.3%) had discordant resistance profiles of clinical significance. Logistic regression analyses indicated that patients on triple therapy were 17.24 times more likely to have concordant drug resistance profile than those on non-triple therapies (OR=17.24, 95% CI: 3.48, 83.33), while patients with increasing number of regimens and years on ART have a decreased rate of concordance (OR = 0.59, 95% CI: 0.32, 1.09 and OR = 0.62, 95% CI: 0.43, 0.88), respectively, than those with less number of regimens and years on ART. Our results show high level of concordance between plasma and PBMC resistance profiles, indicating the possibility of using PBMCs for drug resistance testing in resources-limited settings.
PMCID: PMC3316452  PMID: 22493752
HIV-1 drug resistance; RNA; PBMCs; concordance; discordance; resistance profile; resource-limited setting
9.  Optimization of a Low Cost and Broadly Sensitive Genotyping Assay for HIV-1 Drug Resistance Surveillance and Monitoring in Resource-Limited Settings 
PLoS ONE  2011;6(11):e28184.
Commercially available HIV-1 drug resistance (HIVDR) genotyping assays are expensive and have limitations in detecting non-B subtypes and circulating recombinant forms that are co-circulating in resource-limited settings (RLS). This study aimed to optimize a low cost and broadly sensitive in-house assay in detecting HIVDR mutations in the protease (PR) and reverse transcriptase (RT) regions of pol gene. The overall plasma genotyping sensitivity was 95.8% (N = 96). Compared to the original in-house assay and two commercially available genotyping systems, TRUGENE® and ViroSeq®, the optimized in-house assay showed a nucleotide sequence concordance of 99.3%, 99.6% and 99.1%, respectively. The optimized in-house assay was more sensitive in detecting mixture bases than the original in-house (N = 87, P<0.001) and TRUGENE® and ViroSeq® assays. When the optimized in-house assay was applied to genotype samples collected for HIVDR surveys (N = 230), all 72 (100%) plasma and 69 (95.8%) of the matched dried blood spots (DBS) in the Vietnam transmitted HIVDR survey were genotyped and nucleotide sequence concordance was 98.8%; Testing of treatment-experienced patient plasmas with viral load (VL) ≥ and <3 log10 copies/ml from the Nigeria and Malawi surveys yielded 100% (N = 46) and 78.6% (N = 14) genotyping rates, respectively. Furthermore, all 18 matched DBS stored at room temperature from the Nigeria survey were genotyped. Phylogenetic analysis of the 236 sequences revealed that 43.6% were CRF01_AE, 25.9% subtype C, 13.1% CRF02_AG, 5.1% subtype G, 4.2% subtype B, 2.5% subtype A, 2.1% each subtype F and unclassifiable, 0.4% each CRF06_CPX, CRF07_BC and CRF09_CPX.
The optimized in-house assay is broadly sensitive in genotyping HIV-1 group M viral strains and more sensitive than the original in-house, TRUGENE® and ViroSeq® in detecting mixed viral populations. The broad sensitivity and substantial reagent cost saving make this assay more accessible for RLS where HIVDR surveillance is recommended to minimize the development and transmission of HIVDR.
PMCID: PMC3223235  PMID: 22132237
10.  Development and Application of a Broadly Sensitive Dried-Blood-Spot-Based Genotyping Assay for Global Surveillance of HIV-1 Drug Resistance ▿  
Journal of Clinical Microbiology  2010;48(9):3158-3164.
As antiretroviral therapy (ART) is scaled up in resource-limited countries, surveillance for HIV drug resistance (DR) is vital to ensure sustained effectiveness of first-line ART. We have developed and applied a broadly sensitive dried-blood-spot (DBS)-based genotyping assay for surveillance of HIV-1 DR in international settings. In 2005 and 2006, 171 DBS samples were collected under field conditions from newly diagnosed HIV-1-infected individuals from Malawi (n = 58), Tanzania (n = 60), and China (n =53). In addition, 30 DBS and 40 plasma specimens collected from ART patients in China and Cameroon, respectively, were also tested. Of the 171 DBS analyzed at the protease and RT regions, 149 (87.1%) could be genotyped, including 49 (81.7%) from Tanzania, 47 (88.7%) from China, and 53 (91.4%) from Malawi. Among the 70 ART patient samples analyzed, 100% (30/30) of the Chinese DBS and 90% (36/40) of the Cameroonian plasma specimens were genotyped, including 8 samples with a viral load of <400 copies/ml. The results of phylogenetic analyses indicated that the subtype, circulating recombinant form (CRF), and unique recombinant form (URF) distribution was as follows: 73 strains were subtype C (34%), 37 were subtype B (17.2%), 24 each were CRF01_AE or CRF02_AG (11.2% each), 22 were subtype A1 (10.2%), and 9 were unclassifiable (UC) (4.2%). The remaining samples were minor strains comprised of 6 that were CRF07_BC (2.8%), 5 that were CRF10_CD (2.3%), 3 each that were URF_A1C and CRF08_BC (1.4%), 2 each that were G, URF_BC, and URF_D/UC (0.9%), and 1 each that were subtype F1, subtype F2, and URF_A1D (0.5%). Our results indicate that this broadly sensitive genotyping assay can be used to genotype DBS collected from areas with diverse HIV-1 group M subtypes and CRFs. Thus, the assay is likely to become a useful screening tool in the global resistance surveillance and monitoring of HIV-1 where multiple subtypes and CRFs are found.
PMCID: PMC2937690  PMID: 20660209
12.  Comparative Performance of Several Flexible Docking Programs and Scoring Functions: Enrichment Studies for a Diverse Set of Pharmaceutically Relevant Targets 
Virtual screening by molecular docking has become a widely used approach to lead discovery in the pharmaceutical industry when a high resolution structure of the biological target of interest is available. The performance of three widely-used docking programs (Glide, GOLD, and DOCK) for virtual database screening is studied when they are applied to the same protein target and ligand set. Comparisons of the docking programs and scoring functions using a large and diverse data set of pharmaceutically interesting targets and active compounds are carried out. We focus on the problem of docking and scoring flexible compounds which are sterically capable of docking into a rigid conformation of the receptor. The Glide XP methodology is shown to consistently yield enrichments superior to the two alternative methods, while GOLD outperforms DOCK on average. The study also shows that docking into multiple receptor structures can decrease the docking error in screening a diverse set of active compounds.
PMCID: PMC2547888  PMID: 17585856
13.  Decline in Sulfadoxine-Pyrimethamine-Resistant Alleles after Change in Drug Policy in the Amazon Region of Peru▿ †  
The frequency of alleles with triple mutations conferring sulfadoxine-pyrimethamine (SP) resistance in the Peruvian Amazon Basin has declined (16.9% for dhfr and 0% for dhps compared to 47% for both alleles in 1997) 5 years after SP was replaced as the first-line treatment for Plasmodium falciparum malaria. Microsatellite analysis showed that the dhfr and dhps alleles are of common origin.
PMCID: PMC2224716  PMID: 18025120
14.  Exploring structural variability in X-ray crystallographic models using protein local optimization by torsion-angle sampling 
Torsion-angle sampling, as implemented in the Protein Local Optimization Program (PLOP), is used to generate multiple structurally variable single-conformer models which are in good agreement with X-ray data. An ensemble-refinement approach to differentiate between positional uncertainty and conformational heterogeneity is proposed.
Modeling structural variability is critical for understanding protein function and for modeling reliable targets for in silico docking experiments. Because of the time-intensive nature of manual X-ray crystallographic refinement, automated refinement methods that thoroughly explore conformational space are essential for the systematic construction of structurally variable models. Using five proteins spanning resolutions of 1.0–2.8 Å, it is demonstrated how torsion-angle sampling of backbone and side-chain libraries with filtering against both the chemical energy, using a modern effective potential, and the electron density, coupled with minimization of a reciprocal-space X-ray target function, can generate multiple structurally variable models which fit the X-ray data well. Torsion-angle sampling as implemented in the Protein Local Optimization Program (PLOP) has been used in this work. Models with the lowest R free values are obtained when electrostatic and implicit solvation terms are included in the effective potential. HIV-1 protease, calmodulin and SUMO-conjugating enzyme illustrate how variability in the ensemble of structures captures structural variability that is observed across multiple crystal structures and is linked to functional flexibility at hinge regions and binding interfaces. An ensemble-refinement procedure is proposed to differentiate between variability that is a consequence of physical con­formational heterogeneity and that which reflects uncertainty in the atomic coordinates.
PMCID: PMC2631124  PMID: 18391405
automated refinement; multiple models; conformational heterogeneity; torsion-angle sampling
15.  Pyrosequencing, a High-Throughput Method for Detecting Single Nucleotide Polymorphisms in the Dihydrofolate Reductase and Dihydropteroate Synthetase Genes of Plasmodium falciparum▿  
Journal of Clinical Microbiology  2006;44(11):3900-3910.
A pyrosequencing protocol was developed as a rapid and reliable method to identify the mutations of the dhfr and dhps genes of Plasmodium falciparum that are associated with antifolate resistance. The accuracy and specificity of this method were tested using six laboratory-cultured P. falciparum isolates harboring known single nucleotide polymorphisms (SNPs) in the genes dhfr (codons 50, 51, 59, 108, and 164) and dhps (codons 436, 437, 540, 581, and 613). The lowest threshold for detection of all the SNPs tested by pyrosequencing was the equivalent of two to four parasite genomes. Also, this method was highly specific for P. falciparum, as it did not amplify any DNA products from the other species of human malaria parasites. We also mixed wild-type and mutant-type parasite DNAs in various proportions to determine how pyrosequencing, restriction fragment length polymorphism (RFLP), and direct conventional sequencing (for dhfr) compared with each other in detecting different SNPs in the mixture. In general, pyrosequencing and RFLP showed comparable sensitivities in detecting most of the SNPs in dhfr except for the 164L mutation, which required at least twice the amount of DNA for pyroseqencing as for RFLP. For detecting SNPs in dhps, pyrosequencing was slightly more sensitive than RFLP and direct sequencing. Overall, pyrosequencing was faster and less expensive than either RFLP or direct sequencing. Thus, pyrosequencing is a practical alternative method that can be used in a high-throughput format for molecular surveillance of antimalarial-drug resistance.
PMCID: PMC1698350  PMID: 16957045
16.  Placental Malaria Diminishes Development of Antibody Responses to Plasmodium falciparum Epitopes in Infants Residing in an Area of Western Kenya Where P. falciparum Is Endemic 
To determine the effect of placental malaria (PM) infection on the development of antibody responses to malaria in infants, we measured immunoglobulin G levels to seven different Plasmodium falciparum epitopes by using plasma samples collected at monthly intervals from infants born to mothers with and without PM. Overall, PM was associated with diminished antibody levels to all of the epitopes tested, especially with infants aged ≥4 to 12 months, and the difference was statistically significant for four of the seven epitopes (P < 0.0035). These findings suggest that PM can negatively influence the development of immune responses to malaria in infants.
PMCID: PMC1065200  PMID: 15753250

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