[Advance the construction of "health promotion, prevention, diagnosis, control, treatment, rehabilitation" six-in-one working system of chronic obstructive pulmonary disease in China].

Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease that seriously threatens people's health. It significantly affects the quality of life of patients and presents an overwhelming economic burden on the governmental perspectives, which makes COPD a major public health issue in China. In this paper, we propose some methods that can help to accelerate the implementation of the Healthy China Strategy and promote the change of people's attitudes towards COPD from disease-centered to health-centered. Those methods are composed of many important aspects including the concepts of"population medicine", the improvement of the national health policy for COPD, the consolidation of the original troika strategy of respiratory disciplines and the high-quality implementation of the three major national projects, aiming to inspire people to participate in the six-in-one work system of dealing with COPD encompassing the health promotion, the prevention, the diagnosis, the control, the treatment, and the rehabilitation.

[Classification and imaging diagnosis of Lisfranc joint injuries].

OBJECTIVE: To accelerate the detection rate and accuracy of diagnosis in damage imaging of Lisfranc joint through research on the information of X-ray, CT, and MR imaging of tarsometatarsus joint (also called Lisfranc joint) damage. METHODS: A total of 153 cases of tarsometratisus damage or Lisfranc ligamentous injury patients were chosen during November 2012 to November 2015. Lisfranc injuries were classified according to the Myerson fracture displacements classification and Nunley-Vertullo low-grade injury classification. All the treatment data was performed using SPSS 17.0 software. RESULTS: For Myerson fracture displacements, there were 16 cases in Myerson Ⅰ type (homolateral complete), 100 cases in Myerson Ⅱ type (homolateral incomplete), and 5 cases in Myerson Ⅲ type (divergent). For the low-grade injury, there were 7 cases, 24 cases, and 1 case in Nunley-Vertullo Ⅰ, Ⅱ, Ⅲ type respectively. The probability was 14.9% (18/121) for patients that the initial survey found negative by X-ray imaging diagnosis and was confirmed by subsequent CT or MRI. It was found that the distance between the base of first (M1) and second (M2) metatarsus which was larger than 2 mm was 69.4%(84/121)from the X-ray imaging; there were small chip fractures between the base of M1 and M2 was 47.1% (57/121), and 71.2% (37/52)of small chip fractures in the inside of base of M2 from CT. On MRI, ligament disruption showed the discontinue or normal signal disappearing, and there were 15 cases in the complete disruption condition. CONCLUSION: It should be suggested to take a CT or MRI check for the patients who have highly suspicious Lisfranc injure and the X-ray imaging diagnosis was negative, since there is a certain rate of missed diagnosis for the Lisfranc injure using X-ray imaging. For children and teenagers, the sports injuries and joint strain are common style, such as the injuries caused by jump from higher platform, football/skateboarding injures, etc. If the distance between the base of M1 and M2 is larger than 2 mm or there are small chip fractures between the M1 and M2, the patients would be suspected bone fracture or Lisfranc ligament injure. Myerson Ⅱ type is the most common Lisfranc joint injury , and MRI is best for Lisfranc ligament injury.

Fiber-integrated 780 nm source for visible parametric generation.

We report the development of a fully fiber-integrated pulsed master oscillator power fibre amplifier (MOPFA) source at 780 nm, producing 3.5 W of average power with 410 ps pulses at a repetition rate of 50 MHz. The source consists of an intensity modulated 1560 nm laser diode amplified in an erbium fiber amplifier chain, followed by a fiber coupled periodically poled lithium niobate crystal module for frequency doubling. The source is then used for generating visible light through four-wave mixing in a length of highly nonlinear photonic crystal fiber: 105 mW at 668 nm and 95 mW at 662 nm are obtained, with pump to anti-Stokes conversion slope efficiencies exceeding 6% in both cases.

Prevalence of hepatitis B virus infection among persons with diagnosed diabetes mellitus in the United States, 1999-2010.

The prevalence of hepatitis B virus (HBV) infection among persons with diabetes has not been assessed among the US population, despite increasing reports of HBV transmission in institutional care settings. Using national survey data, we found a 60% higher prevalence of HBV infection among persons with (vs without) diagnosed diabetes.

Comparison of antioxidant activities of different parts from snow chrysanthemum (Coreopsis tinctoria Nutt.) and identification of their natural antioxidants using high performance liquid chromatography coupled with diode array detection and mass spectrometry and 2,2'-azinobis(3-ethylbenzthiazoline-sulfonic acid)diammonium salt-based assay.

Snow chrysanthemum (Coreopsis tinctoria Nutt.), a world-widely well-known flower tea material, has attracted more and more attention because of its beneficial health effects such as antioxidant activity and special flavor. In this study, a high performance liquid chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD-MS) and 2,2'-azinobis(3-ethylbenzthiazoline-sulfonic acid)diammonium salt (ABTS) based assay was employed for comparison and identification of antioxidants in different samples of snow chrysanthemum. The results showed that snow chrysanthemum flowers possessed the highest while stems presented the lowest antioxidant capacities. Fourteen detected peaks with antioxidant activity were temporarily identified as 3,4',5,6,7-pentahydroxyflavanone-O-hexoside, chlorogenic acid, 2R-3',4',8-trihydroxyflavanone-7-O-glucoside, flavanomarein, flavanocorepsin, flavanokanin, quercetagitin-7-O-glucoside, 3',5,5',7-tetrahydroxyflavanone-O-hexoside, marein, maritimein, 1,3-dicaffeoylquinic acid, coreopsin, okanin and acetyl-marein by comparing their UV spectra, retention times and MS data with standards or literature data. Antioxidants existed in snow chrysanthemum are quite different from those reported in Chrysanthemum morifolium, a well-known traditional beverage in China, which indicated that snow chrysanthemum may be a promising herbal tea material with obvious antioxidant activity.

The expanding regional diversity of the acquired immunodeficiency syndrome epidemic in the United States.

BACKGROUND: The geographic spread of the human immunodeficiency virus (HIV) epidemic reflects multiple subepidemics in different regions and population groups. METHODS: To describe regional trends in the acquired immunodeficiency syndrome (AIDS) in the United States, we analyzed national surveillance data for persons with AIDS diagnosed from 1988 through 1991. RESULTS: Highest annual AIDS incidence rates were in the US territories (52.7 per 100,000) and the Northeast (27.7 per 100,000). The greatest percentage increases were in the US territories (68.8%), the South (60.1%), and the Midwest (52.4%). Men who have sex with men constituted the majority of AIDS cases nationally (54.6%), as well as in the Midwest (67.8%), the South (57.4%), and the West (75.3%). Among injecting drug users, the greatest rates of increase in AIDS cases were observed among blacks in the South. Although large increases in the number of persons with HIV transmitted through heterosexual contact were reported from almost all regions, the largest increase was in the South. CONCLUSION: High rates of increase in AIDS cases from the Midwest, South, and US territories probably reflect later entry of HIV into these regions compared with the earlier HIV epidemics in large metropolitan areas of the Northeast and West. In particular, because the South has the largest population of the regions, and sexually transmitted disease surveillance data suggest that substantial populations in the South are at risk, the marked increase in AIDS incidence in this region suggests that the major impact of the epidemic may yet be seen. The continuing spread of HIV and AIDS in different communities and regions demonstrates the need to expand preventive and therapeutic services.

Decoding glycome of Astragalus membranaceus based on pressurized liquid extraction, microwave-assisted hydrolysis and chromatographic analysis.

Carbohydrates in herbs are a relatively untapped source of new drugs and health beneficial ingredients. Their analysis has been developed as a novel aspect in quality control and herbal glycomics. In this study, glycome of Astragalus membranaceus was decoded based on optimized pressurized liquid extraction (PLE), microwave-assisted acidic hydrolysis (MAAH) and comprehensive chromatographic approaches. Twelve saccharides including sucrose, galacturonic acid, mannitol, fructose, rhamnose, ribose, arabinose, fucose, xylose, mannose, glucose and galactose were quantitatively analyzed by GC-MS and HPLC-CAD (charged aerosol detectors). Different columns, including Prevail Carbohydrate ES, XBridge Amide and CARBOSep CHO-820 CA for HPLC-CAD analysis, were compared for evaluation of oligosaccharides. The polysaccharides in water extract of Astragalus membranaceus were characterized by high performance size exclusive chromatography (HPSEC) combined with multiple angle light scattering detection (MALSD) and refractive index detection (RID). The results showed that A. membranaceus contained more than 108.5mgg(-1) free sucrose and small amounts of glucose 9.6-26.0mgg(-1) and fructose 8.7-22.9mgg(-1). While its polymeric carbohydrates were composed of glucose 71.0-162.3mgg(-1), galacturonic acid 52.0-113.4mgg(-1), arabinose 22.8-54.4mgg(-1) and small amounts of galactose, rhamnose, xylose and mannose. CARBOSep CHO-820 CA showed its potential in simultaneously analyzing oligosaccharides and uronic acid, especially only the environment-friendly water mobile phase was used. HPSEC-MALSD-RID showed that there were three different molecular weight distributions of polysaccharides in A. membranaceus and the average molecular weight were 21901.1, 2038.5, and 353.4kDa. Hierarchical clustering analysis and principal component analysis demonstrated that A. membranaceus from different regions showed variations both in free and polymeric carbohydrates, which indicated that carbohydrates should be evaluated for the proper quality control of A. membranaceus. Rha, Ara, Xyl, Man and Gal were found to be the main elements for quality evaluation of polymeric carbohydrates in A. membranaceus by factor analysis. The strategy for decoding the glycome based on chromatographic approaches including GC-MS, HPLC-CAD and HPSEC-MALSD-RID after pressurized liquid extraction and microwave-assisted hydrolysis could be applied for carbohydrates profiling in herbs and beneficial for their quality control.

Income and AIDS rates in Los Angeles County.

OBJECTIVE: To examine the relationship between income and AIDS rates in Los Angeles County (LAC) by race/ethnicity. METHODS: 1990 US census data were used to classify LAC postal zones (zip codes) by median household income into low-, middle-, and high-income strata. AIDS rates were calculated for each income stratum based on 15,805 AIDS cases diagnosed from 1987 through 1992 and reported to the county health department. RESULTS: The AIDS rate was highest among residents of low-income areas (252.8 per 100,000), intermediate among residents of middle-income areas (161.2 per 100,000), and lowest among residents of high-income areas (82.0 per 100,000). This trend in rates was present in all racial/ethnic groups examined and was most pronounced among whites (675.1, 226.7, and 88.4 per 100,000, respectively). Residents of low-income areas accounted for 78% of AIDS cases among blacks, 67% among Hispanics, and 47% among whites. CONCLUSIONS: These findings suggest a strong inverse relationship between income and AIDS rates in LAC that is consistent across racial/ethnic groups. Prevention programs and treatment services should be directed most intensively to low-income neighborhoods in this county.

Surveillance of AIDS-defining conditions in the United States. Adult/Adolescent Spectrum of HIV Disease Project Group.

OBJECTIVE: To examine the reporting of AIDS-defining illnesses using two national surveillance systems. METHODS: Comparison of AIDS indicator diseases reported to the national AIDS reporting system (ARS) for AIDS cases diagnosed from January 1990-December 1992 among individuals aged > 13 years in 10 metropolitan areas, with that observed in the Adult/Adolescent Spectrum of HIV Disease (ASD) project, a surveillance project that monitors the clinical diagnoses of HIV-infected individuals receiving medical care. RESULTS: In the 10 metropolitan areas, 39,265 individuals with AIDS were reported to ARS, and 5969 with AIDS had medical record reviews as part of ASD. At initial AIDS diagnosis, the number of indicator diseases reported to ARS was almost identical to the number observed in ASD (mean number of diagnoses, ARS 1.3; ASD 1.2). However, ASD recorded a greater number of diagnoses over time than ARS (mean number of indicator diagnoses > 12 months after initial diagnosis, ASD 2.3; ARS 1.4). Conditions that typically occur late in the course of AIDS such as Mycobacterium avium infection and cytomegalovirus disease, were more frequently recorded by ASD than by ARS. CONCLUSION: ARS provides complete, population-based information on the frequency of AIDS-defining conditions at initial diagnosis. However, specialized surveillance projects such as ASD are needed to accurately describe subsequent AIDS-defining conditions.

Clinical disease associated with HIV-1 subtype B' and E infection among 2104 patients in Thailand.

BACKGROUND: Two HIV-1 envelope subtypes have accounted for virtually all infections in Thailand: subtype B' (Thai B), found mainly in injection drug users (IDU), and subtype E, found in over 90% of sexually infected persons and an increasing proportion of IDU in recent years. It remains unclear whether there are differences in pathogenesis associated with these HIV-1 subtypes. METHODS: From November 1993 to June 1996, demographic, risk, clinical, and laboratory data were collected by enhanced surveillance from HIV-infected inpatients (> or =14 years) at an infectious disease hospital near Bangkok. HIV-1 subtype was determined by V3-loop peptide enzyme immunoassay (EIA). Because of confounding, multivariate analyses were stratified by risk category and controlled for sex and age. RESULTS: The infecting HIV-1 subtype was determined for 2104 (94.9%) of 2217 HIV-infected patients with complete data: 284 (13.5%) were subtype B', 1820 (86.5%) were E. Specimens from 113 (5.1%) patients were non-reactive or dually reactive on peptide EIA and were excluded. Among IDU, 199 (67.2%) had subtype B', and 97 (32.7%) had E. IDU accounted for 70.1% (199/284) of patients with subtype B' and 5.3% (97/1820) of those with E. Patients infected with HIV-1 subtypes B' or E had similar spectrums of opportunistic infections (OI), levels of immunosuppression, and in-hospital mortality rates. Of patients who did not inject drugs, more patients infected with subtype E had extrapulmonary cryptococcosis than those with subtype B' (adjusted odds ratio, 2.6; 95% confidence interval, 1.28-5.37). CONCLUSION: HIV-1 subtypes B' and E seem to be associated with similar degrees of immunosuppression and, with one exception, with similar OI patterns. These data do not suggest an association between HIV-1 subtype and differences in pathogenicity.

HIV risk behaviors but absence of infection among drug users in detoxification centers outside Yunnan province, China, 1993.

OBJECTIVES: To date, HIV spread in China has occurred principally among injecting drug users (IDU) in remote border regions of Yunnan province. We therefore sought to better understand the risks for and prevalence of HIV infection among drug users in parts of China outside Yunnan province. METHODS: A behavioral survey of drug use and AIDS-related knowledge was conducted among all consenting drug users who entered treatment from 1 November to 31 December 1993 in seven provincial drug detoxification centers outside Yunnan province. After giving verbal informed consent, all drug users were tested for HIV. RESULTS: Of the 1293 study participants, 207 (16%) reported injecting drugs. The proportion of IDU among all drug users varied widely by region, from 1% in Sha'anxi and Gansu provinces in the northwest region to 58% in Guangxi province in the south. IDU were more likely than non-IDU to be single and unemployed or self-employed, but did not differ in other demographic aspects. Among all drug users, 2% reported sharing needles without cleaning equipment, while 5% shared with some cleaning. Although 1060 (82%) drug users had heard of AIDS and most knew about its sexual (79%), parenteral (77%), and perinatal (60%) modes of transmission, many had misconceptions about its spread by casual contact. Of the 207 IDU tested for HIV, none were HIV-positive (95% confidence interval, 0--1.4). CONCLUSION: The absence of HIV infections detected in this study suggests that the prevalence of HIV is currently low among IDU in China outside the Yunnan province. However, the behavior of these IDU puts them at high risk for HIV infection. Prevention efforts are needed to prevent the spread of HIV among IDU throughout China and to avoid the experience of neighboring countries in Asia.

PIP: Representatives of the World Bank (RWB) claim that a published argument linking some of their policies to the development of a situation which fosters the spread of AIDS can be summarized as claiming that "economic development is bad because it facilitates the spread of communicable disease by bringing people into closer and more frequent contact." The RWB acknowledge that economic development facilitates the spread of infection but defend economic development because it also provides the knowledge and means to combat disease. The RWB claim that the logical conclusion of the alternative development strategy proposed by their critics would mean a return to the stone age. The RWB further accuse their critics of ignoring evidence that countries undergoing structural adjustment have achieved faster economic growth, a reduction in poverty, and an increase in health spending. The critics are charged with failing to ask whether the criticisms leveled at the World Bank's treatment of AIDS as a public health priority are valid, with failing to point out that the labor migration in south Africa which incubated AIDS is older than structural adjustment, and with failure to note that Brazil's avoidance of economic equilibrium during the 1980s impoverished the country. Finally, the RWB charged that their critics disregard economics, public health, and the scientific method in their attempt to make AIDS the only thing that matters.

HIV infection and breast-feeding: policy implications through a decision analysis model.

OBJECTIVES: (1) To develop a comprehensive decision analysis model to compare mortality associated with HIV transmission from breast-feeding with the mortality from not breast-feeding in different populations and (2) to perform sensitivity analyses to illustrate critical boundaries for guiding research and policy. METHODS: Using a decision tree, mortality rates were estimated for all children, children born to mothers infected during pregnancy, and children born to mothers who were uninfected at delivery. Given various assumptions about child mortality rates, relative risks of mortality among children who are not breast-fed compared with those who are (R), rates of HIV transmission from breast-feeding, HIV prevalence, and HIV incidence, scenarios were created and sensitivity analysis used to delineate critical boundaries. RESULTS: Our model shows that only in situations where R is approximately < or = 1.5 and HIV incidence/prevalence is high (prevalence > 10%, incidence > 5%) would universal breast-feeding result in equal or higher mortality compared with non-breast-feeding. Among populations in many developing countries, where there is a high relative risk of mortality if breast-feeding is not practiced, if R > 3, overall mortality is almost always lower among children who are breast-fed, even by HIV-infected mothers. In situations where maternal HIV status is known, the decision whether to breast-feed is largely dependent on the magnitude of additional mortality risk if the child is not breast-fed. The model illustrates the importance of distinguishing between population and individual recommendations. CONCLUSIONS: Based on available data, the model supports current World Health Organization and Centers for Disease Control recommendations on HIV infection and breast-feeding. Given the importance of breast-feeding and the global impact of HIV infection, more research is needed, especially to clarify the range of HIV transmission rates from breast-feeding and to expand specific assessments of relative risks for different areas of the world.

PIP: HIV/AIDS specialists have developed and applied 3 different scenarios to a comprehensive decision analysis model to estimate mortality rates for children of mothers infected with HIV during pregnancy and for children of mothers who were not infected with HIV during delivery. Scenario I represents Central Africa where HIV prevalence and incidence are high. Some scenario I assumptions are HIV prevalence in pregnant women of 30% and proportion of initially uninfected women who become infected after delivery during lactation (d) of 6%. Scenario II is a population where HIV epidemic is rather recent (e.g., some parts of Asia). Its assumptions are HIV prevalence of 5%, and s is 2%. Scenario III symbolizes high-risk populations in North America and Western Europe (HIV prevalence and s = 1%). The scenarios also consider child mortality rates and relative risks (RRs) of mortality of breast fed children and those who were not breast fed. Universal breast feeding would effect equal or higher mortality than non-breast feeding, when the RR of mortality is no more than 1.5 and HIV prevalence/incidence is high (high prevalence = 10% and high incidence = 5%). In developing countries, where the RR of mortality is high if children are not breast fed (RR 3), breast fed children have almost always lower child mortality than those who are not breast fed, regardless of HIV infection status. The decision to breast feed when the HIV status is known depends greatly on the degree of an additional mortality risk if an infant is not breast fed. The model substantiates WHO and CDC recommendations: HIV-positive women in the UK and the US should not breast feed, while those in developing countries with high RR of child mortality should breast feed. Additional research would define the range of HIV transmission rates from breast feeding and increase specific assessments of RRs for various parts of the world.

Protease sequences from HIV-1 group M subtypes A-H reveal distinct amino acid mutation patterns associated with protease resistance in protease inhibitor-naive individuals worldwide. HIV Variant Working Group.

BACKGROUND: Although numerous mutations that confer resistance to protease inhibitors (PRI) have been mapped for HIV-1 subtype B, little is known about such substitutions for the non-B viruses, which globally cause the most infections. OBJECTIVES: To determine the prevalence of PRI-associated mutations in PRI-naive individuals worldwide. DESIGN: Using the polymerase chain reaction, protease sequences were amplified from 301 individuals infected with HIV-1 subtypes A (79), B (95), B' (19), C (12), D (26), A/E (23), F (26), A/G (11), and H (3) and unclassifiable HIV-1 (7). Amplified DNA was directly sequenced and translated to amino acids to analyze PRI-associated major and accessory mutations. RESULTS: Of the 301 sequences, 85% contained at least one codon change giving substitution at 10, 20, 30, 36, 46, 63, 71, 77, or 82 associated with PRI resistance; the frequency of these substitutions was higher among non-B (91%) than B (75%) viruses (P < 0.0005). Of these, 25% carried dual and triple substitutions. Two major drug resistance-conferring mutations, either 20M or 30N, were identified in only three specimens, whereas drug resistance accessory mutations were found in 252 isolates. These mutations gave distinct prevalence patterns for subtype B, 63P (62%) > 77I (19%) > 10I/V/R (6%) = 361 (6%) = 71T/V (6%) > 20R (2%), and non-B strains, 36I (83%) > 63P (17%) > 10I/V/R (13%) > 20R (10%) > 77I (2%), which differed statistically at positions 20, 36, 63, 71, and 77. CONCLUSIONS: The high prevalence of PRI-associated substitutions represent natural polymorphisms occurring in PRI-naive patients infected with HIV-1 strains of subtypes A-H. The significance of distinct mutation patterns identified for subtype B and non-B strains warrants further clinical evaluation. A global HIV-1 protease database is fundamental for the investigation of novel PRI.

Viral load differences in early infection with two HIV-1 subtypes.

OBJECTIVES: Information on early HIV-1 infection has come primarily from studies of persons infected with subtype B in North America and Europe; much less is known about other subtypes. The purpose of the present study was to compare the virologic and immunologic parameters following seroconversion among recently-infected persons infected with either of two different HIV-1 subtypes. METHOD: A prospective cohort study was carried out at methadone treatment clinics administered by the Bangkok Metropolitan Administration, Thailand. A total of 130 HIV-1-infected seroconverters (103 with HIV-1 subtype E and 27 with subtype B) were included in the study. The main outcome measures were serial HIV-1 RNA viral load, natural killer cell percentage, CD4 and CD8 lymphocyte counts since seroconversion. RESULTS: The demographic and behavioral characteristics of persons with either subtype were similar. Median RNA viral levels at the earliest time within 3 months of seroconversion were more than three times higher for persons infected with subtype E than subtype B (63 100 versus 18 050 copies/ml, P = 0.001). However, this difference decreased over time such that viral loads were similar at 12, 18, and 24 months following seroconversion. The CD4 and CD8 lymphocyte counts were similar in infections with either subtype during the entire period up to 24 months post-seroconversion. CONCLUSIONS: Higher viral loads associated with subtype E may result from inter-subtype biological differences; however, the epidemiological dynamics of transmission in Bangkok may have also contributed to this phenomenon.

A molecular epidemiologic survey of HIV in Uganda. HIV Variant Working Group.

OBJECTIVE: Previous data, based on a small sampling of convenience, reported subtypes A, B, C, D, and G in Uganda, but neither the extent nor the proportion of these subtypes could be evaluated. To establish correctly the prevalence and distribution of HIV-1 subtypes, we analysed viral clades in 739 HIV-1-seropositive specimens from different areas of Uganda. METHODS: Blood specimens from 1100 patients were collected in five districts of Uganda. Within this collection, 929 HIV-1-seroreactive samples underwent analysis of viral DNA, and 739 were selected for further subtyping in env or pol regions. RESULTS: Using a combination of subtype A- and D-specific probes to C2-V3 region and DNA sequencing, HIV-1 env subtypes were determined in 594 specimens: 341 were of subtype A (57.4%), 250 of subtype D (42.1%), and three of subtype C (0.5%). Sixty-two samples showed reactivity with both probes, suggesting potential mixed infections, cross-reactivity to probes, or possibly other subtypes. Subsequent sequence analysis of 19 randomly selected specimens revealed subtypes A (n = 4), D (n = 12), and C (n = 3). Sequence analysis of the 27 samples chosen from the remaining 83 samples, which could be amplified only with viral gp41 or protease gene primers, classified them as subtypes A (n = 13) and D (n = 14). No significant clinical, demographic, or geographic differences were found between HIV-1 infections with viruses of subtypes A and D, despite considerable genetic diversity within these clades. CONCLUSIONS: This is the first major population-based study of the prevalent HIV-1 strains in an African country selected for vaccine trials. The subtyping methods we describe should be of use to investigators seeking to conduct large-scale screening for HIV variants in other populations.

Impact of HIV type 1 subtype variation on viral RNA quantitation.

We evaluated the performance of three HIV-1 RNA quantitation methods (Amplicor HIV-1 MONITOR-1.0, NASBA, and Quantiplex HIV RNA 2.0 [branched DNA (bDNA)]) using plasma specimens (N = 60) from individuals from Asia and Africa infected with one of three HIV-1 subtypes (A, Thai B [B'] or E; N = 20 each). Our results demonstrate that of the 20 subtype A specimens, 19 were quantifiable by the bDNA assay compared with 15 by the MONITOR-1.0 and 13 by NASBA. Of those quantifiable, the mean log10 difference was 0.93 between bDNA and MONITOR-1.0 and 0.46 between bDNA and NASBA. For subtype B' specimens, the correlation among methods was better with only 2 specimens missed by NASBA and 3 by the bDNA assay. However the missed specimens had viral burden near the lower limit (1000 copies/ml) for these assays. For the 20 subtype E specimens, MONITOR-1.0 and NASBA quantified RNA in 17 and 14 specimens, respectively, as compared with 19 specimens quantified by the bDNA assay. The correlation among different assays, especially between bDNA/NASBA and MONITOR-1.0/NASBA, was poor, although the mean log10 difference for subtype E specimens was 0.4 between bDNA and MONITOR-1.0 and only 0.08 between bDNA and NASBA. The addition of a new primer set, designed for non-B HIV-1 subtypes, to the existing MONITOR assay (MONITOR-1.0+) resulted in RNA detection in all 60 specimens and significantly improved the efficiency of quantitation for subtypes A and E. Our data indicate that HIV-1 subtype variation can have a major influence on viral load quantitation by different methods. Periodic evaluation and modification of these quantitative methods may be necessary to ensure reliable quantification of divergent viruses.

A comparison of full-length glycoprotein 120 from incident HIV type 1 subtype E and B infections in Bangkok injecting drug users with prototype E and B strains that are components of a candidate vaccine.

Complete gp120 sequence information was obtained from eight persons with incident HIV-1 infections (four subtype E and four subtype B) who were part of a prospective injecting drug user (IDU) cohort in Bangkok, Thailand, during 1996-1998. The incident subtype E strains were similar to the prototype subtype E strain CM244 isolated in 1992 in northern Thailand. The incident subtype B strains displayed divergence, in both overall genetic distance and other significant gp120 characteristics, from the prototype North American subtype B strain HIV-MN. Recombinant gp120s derived from CM244 and HIV-MN strains are components of a vaccine that is undergoing phase III efficacy testing, begun in March 1999, among Bangkok area IDUs. The information presented here will be important in the evaluation of any breakthrough HIV-1 infections occurring among vaccinees during the vaccine trial and in ongoing vaccine development efforts in Thailand.

Genetic characterization and phylogenetic analysis of HIV-1 subtype C from Uganda.

To better understand the emergence of subtype C and its potential impact on vaccine efforts in Uganda, we have characterized subtype C sequences from Uganda (n = 13), Zimbabwe (n = 11), Mozambique (n = 5), South Africa (n = 4), and India (n = 3). Phylogenetic analysis of subtype C sequences in the env gp41 gene region revealed multiple subclusters within subtype C. Further, while most Ugandan specimen subclustered together, other subclusters did not reflect a clear geographic location. The nucleotide divergence within the Ugandan subset was 8.2% (6.1-9.8%) compared with 9.5% (2.5-15%) for the other subtype C gp41 sequences. The protein sequence alignment revealed marked sequence conservation of major immunodominant epitopes within the gp41 region.

Evaluation of a sensitive/less-sensitive testing algorithm using the 3A11-LS assay for detecting recent HIV seroconversion among individuals with HIV-1 subtype B or E infection in Thailand.

The development of a serologic algorithm to determine recent HIV seroconversion, using sensitive/less-sensitive testing strategies, has generated widespread interest in applying this approach to estimate HIV-1 incidence in various populations around the world. To evaluate this approach in non-B subtypes, longitudinal specimens (n = 522) collected from 90 incident infections among injecting drug users in Bangkok (subtype B infection, n = 18; subtype E infection, n = 72) were tested by the 3A11-LS assay. Standardized optical density (SOD) was calculated, using median values, and the window period between seroconversion as determined by sensitive and less sensitive tests was estimated by a maximum-likelihood model described previously. Our results show that the mean window period of the 3A11-LS assay was 155 days (95% CI, 128-189 days) for subtype B but was 270 days (95% CI, 187-349 days) for subtype E specimens from Thailand. About 4% of individuals with incident subtype E infections remained below the threshold (SOD of 0.75), even 2 years after seroconversion. Among the patients with clinical AIDS and declining antibodies, none of the 7 individuals with subtype B, but 10 (8.7%) of 115 with subtype E infections, were misclassified as recent infections. Lowering the cutoff to an SOD of 0.45 for subtype E specimens resulted in a mean window period of 185 days (95% CI, 154-211 days), with all individuals seroconverting, and reduced the number of subtype E-infected patients with AIDS who were misclassified as having recent infection to 2.6%. Our results demonstrate that the 3A11-LS assay has different performance characteristics in detecting recent infections among individuals infected with subtypes B or E. Determining appropriate cutoffs and mean window periods for other HIV-1 subtypes will be necessary before this approach can be reliably implemented in settings where non-B subtypes are common.

The development and evaluation of a probe hybridization method for subtyping HIV type 1 infection in Uganda.

We developed a method for large-scale screening of HIV-1 genotypic variation based on DNA probe hybridization. Nested PCR amplifications were performed to generate fragments in the env C2-V3 region and also in the gp41 region, which encompasses the immunodominant domain. The proviral DNA sequences were derived from 68 samples and phylogenetically analyzed. For comparison, the C2-V3 fragment was used in DNA probe hybridization to rapidly determine the infecting HIV subtype. The hybridizing probes were designed on the basis of the two most prevalent subtypes in Uganda, A and D. The results were compared to evaluate the feasibility of using this hybridization method for large-scale genotypic screening. Sequence analysis of the 68 amplified PCR fragments showed that 39 were subtype A and 29 were subtype D. The results of DNA hybridization to the amplified products with A and D subtype-specific probes were more than 90% concordant with the subtypes determined by sequence analysis. Our findings suggest that probe hybridization with subtype-specific probes is effective for large-scale screening of HIV-infected populations. Application of this method will significantly reduce the time needed for large, population-based investigations.