[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.

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.

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.

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.

Preparation of inulin-type fructooligosaccharides using fast protein liquid chromatography coupled with refractive index detection.

A fast protein liquid chromatography coupled with refractive index detection (FPLC-RID) method was firstly developed for preparation and purification of fructooligosaccharides with different degree of polymerization from burdock, Arctium lappa. After extraction with 60% ethanol and decolorization with MCI gel CHP20P, total fructooligosaccharides were purified on Bio-Gel P-2 column eluted with water at the flow rate of 0.3 ml/min, which was the optimized conditions. The obtained fructooligosaccharides with degree of polymerization of 3-9 were identified based on their methylation analysis, MS and NMR data. This method has the advantages of high automation, good recovery and easy performance, which could be used for preparation of FOS from other sources, as well as other targeted compounds without UV absorbance.

A novel strategy with standardized reference extract qualification and single compound quantitative evaluation for quality control of Panax notoginseng used as a functional food.

Root of Panax notoginseng (Burk.) F.H. Chen (Sanqi in Chinese) is one of traditional Chinese medicines (TCMs) based functional food. Saponins are the major bioactive components. The shortage of reference compounds or chemical standards is one of the main bottlenecks for quality control of TCMs. A novel strategy, i.e. standardized reference extract based qualification and single calibrated components directly quantitative estimation of multiple analytes, was proposed to easily and effectively control the quality of natural functional foods such as Sanqi. The feasibility and credibility of this methodology were also assessed with a developed fast HPLC method. Five saponins, including ginsenoside Rg1, Re, Rb1, Rd and notoginsenoside R1 were rapidly separated using a conventional HPLC in 20 min. The quantification method was also compared with individual calibration curve method. The strategy is feasible and credible, which is easily and effectively adapted for improving the quality control of natural functional foods such as Sanqi.

Viral hepatitis C gets personal--the value of human genomics to public health.

About 180 million people worldwide are chronically infected with hepatitis C virus (HCV), with 3-4 million newly infected each year. Only 15-25% of acute HCV infections clear spontaneously, and the remainder persists as chronic HCV infection. More than 350,000 people die every year from hepatitis C-related liver failure and cancer. There is currently no vaccine and the standard-of-care therapies--peg-interferon alpha (pegIFN) plus ribavirin (RBV)--are expensive and have serious side effects. Also, they may be effective in only 40-50% of patients infected with HCV genotype 1, the most common HCV genotype in the US. Interleukin 28B (IL28B) genotype was recently and convincingly associated with response to pegIFN and RBV therapy. It has emerged as a robust pretreatment predictor of sustained virological response (SVR, i.e. virologic clearance) to pegIFN and RBV as well as to new triple therapy regimens that include a direct-acting antiviral agent with pegIFN and RBV and increase SVR rates as much as 75% in patients infected with HCV genotype 1. Testing for IL28B genotype may contribute to clinical decision-making and could inform clinical guidelines and public health policies.

Quality evaluation of lentinan injection produced in China.

Lentinan is a ß-(1→6) branched ß-(1→3)-glucan derived from Lentinula edodes mushroom. It has been developed as an adjuvant to cancer chemotherapy and clinically used in Japan and China. Up to date, six lentinan injections or powders for injection produced by individual manufacturers were widely used in clinic in China. Since antitumor effect of lentinan is highly related to its structure, conformation and molecular weight, quality evaluation of lentinan injection produced in China is crucial to ensure its efficacy, which has never been performed. In this study, absolute molecular weights and their distributions of lentinan in multiple batches of injections from different manufacturers in China were firstly determined through a high performance size exclusive chromatography (HPSEC) combined with multiple angle light scattering detection (MALSD) and refractive index detection (RID). Enzymatic (ß-1,3-D-glucanase) digestion characters and triple helical conformation of lentinan were also investigated. The results showed that quality consistency of lentinan injections from different manufacturers in China was poor, which should be greatly improved to ensure their safety and efficacy.

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.

Contrast-free MRA at 3.0 T for the detection of intracranial aneurysms.

OBJECTIVE: We prospectively investigated the diagnostic accuracy of contrast-free 3-dimensional time-of-flight magnetic resonance angiography (3D-TOF-MRA) with volume rendering (VR) at 3.0 T to detect intracranial aneurysms in a large cohort of patients. METHODS: We conducted a prospective clinical study including 411 patients with suspected aneurysms and other cerebral vascular diseases who were referred for contrast-free 3D-TOF-MRA at 3.0 T prior to digital subtraction angiography (DSA). 2D-DSA and VR-DSA were regarded as the gold standard. Forty-two patients were excluded. Accuracy, sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) as measures to detect or rule out intracranial aneurysms were determined by patient-, aneurysm-, vessel-, and aneurysm size-based evaluations. RESULTS: In all 369 patients investigated, VR-DSA revealed 307 aneurysms in 246 patients (66.7%) and no aneurysm in 123 patients. The patient-based evaluation by VR 3D-TOF-MRA at 3.0 T yielded an accuracy of 97.6%, a sensitivity of 99.2%, specificity of 94.4%, PPV of 97.2%, and NPV of 98.3% in the detection of intracranial aneurysms. The aneurysm-based evaluation yielded an accuracy of 98.3%, sensitivity of 99.3%, specificity of 96.9%, PPV of 97.8%, and NPV of 99.1%. The vessel-based evaluation yielded accuracy of 98.8%, sensitivity of 99.2%, specificity of 98.5%, PPV of 97.5%, and NPV of 99.6%. The evaluation based on aneurysm sizes yielded similar results. CONCLUSIONS: VR 3D-TOF-MRA at 3.0 T accurately identified the presence of intracranial aneurysms. High PPV and NPV indicated that VR 3D-TOF-MRA at 3.0 T may replace DSA as a contrast-free, noninvasive, and non-radiation-based modality for the diagnosis and screening of intracranial aneurysms.

Determination of nucleotides, nucleosides and their transformation products in Cordyceps by ion-pairing reversed-phase liquid chromatography-mass spectrometry.

An ion-pairing reversed-phase liquid chromatography-mass spectrometry (IP-RP-LC-MS) was developed for the determination of nucleotides, nucleosides and their transformation products in Cordyceps. Perfluorinated carboxylic acid, namely pentadecafluorooctanoic acid (PDFOA, 0.25mM), was used as volatile ion-paring agent and a reversed-phase column (Agilent ZORBAX SB-Aq column) was used for the separation of three nucleotides namely uridine-5'-monophosphate (UMP, 0.638-10.200microg/mL), adenosine-5'-monophosphate (AMP, 0.24-7.80microg/mL) and guanosine-5'-monophosphate (GMP, 0.42-13.50microg/mL), seven nucleosides including adenosine (0.55-8.85microg/mL), guanosine (0.42-6.75microg/mL), uridine (0.33-10.50micro/mL), inosine (0.21-6.60microg/mL), cytidine (0.48-15.30microg/mL), thymidine (0.20-6.30microg/mL) and cordycepin (0.09-1.50microg/mL), as well as six nucleobases, adenine (0.22-6.90microg/mL), guanine (0.26-4.20microg/mL), uracil (0.38-12.15microg/mL), hypoxanthine (0.13-4.20microg/mL), cytosine (0.39-12.45microg/mL) and thymine (0.26-8.25microg/mL) with 5-chlorocytosine arabinoside as the internal standard. The overall LODs and LOQs were between 0.01-0.16microg/mL and 0.04-0.41microg/mL for the 16 analytes, respectively. The contents of 16 investigated compounds in natural and cultured Cordyceps were also determined and compared after validation of the developed IP-RP-LC-MS method. The transformations of nucleotides and nucleosides in Cordyceps were evaluated based on the quantification of the investigated compounds in three extracts, including boiling water extraction (BWE), 24h ambient temperature water immersion (ATWE) and 56h ATWE extracts. Two transformation pathways including UMP-->uridine-->uracil and GMP-->guanosine-->guanine were proposed in both natural Cordyceps sinensis and cultured Cordyceps militaris. The pathway of AMP-->adenosine-->inosine-->hypoxanthine was proposed in natural C. sinensis, while AMP-->adenosine-->adenine in cultured C. militaris. However, the transformation of nucleotides and nucleosides was not found in commercial cultured C. sinensis.

Random amplified polymorphic DNA (RAPD) analysis and the nucleosides assessment of fungal strains isolated from natural Cordyceps sinensis.

Random amplified polymorphic DNA (RAPD) and high performance liquid chromatography (HPLC) were applied to investigate genetic and chemical variations of 2 natural C. sinensis, 16 fungal strains isolated from C. sinensis, and 2 fungal strains of C. militaris. Five of the 68 arbitrary decamer primers were available for discrimination of the investigated samples. As a result, 20 investigated samples were divided into three main clusters according to the genetic distance, and some fungal strains isolated from natural C. sinensis were obviously different. But according to the contents of nucleosides, including uracil, uridine, hypoxanthine, inosine, guanosine, adenosine, adenine, and cordycepin, natural and cultured Cordyceps were in two individual sub-groups, which suggested that chemical characteristics among cultured mycelia of different fungal strains isolated from natural C. sinensis were similar, but they were different from natural one.

Establishment of an experimental intracranial internal carotid artery model and the application in covered-stent navigability testing.

BACKGROUND AND PURPOSE: The carotid siphon is a natural barrier to intracranial interventions. Our aim was to make a model of the human intracranial internal carotid artery (ICA) and to test the navigability of covered stents for intracranial applications. MATERIALS AND METHODS: A digital tube was made on the basis of raw MR images of the human ICA. It was transferred into 10 physical models and then coated with silicone by using a 3D rapid prototyping (RP) machine. Ten dogs then underwent surgery. Their common carotid arteries (CCAs) were exposed, cut, and passed through 1 of the tubes. Finally, the vascular models were made by reanastomosis of their CCAs. Eight expended polytetrafluoroethylene (e-PTFE) covered stents (two 3.5 x 16 mm, two 3.5 x 13 mm, two 3.5 x 10 mm, and two 3.5 x 7 mm) were implanted 1 week later. Two dogs remained as controls. The performance of the device was evaluated by angiography and histopathologic examination. RESULTS: Ten animal models were successfully constructed. There was no vascular spasm or thrombosis when assessed by angiography. Destruction of the tunica intima and media was found in the 3.5 x 16 mm stent group. Destruction of the endothelium was found in the 3.5 x 13 mm stent group, and only flattening of the endothelium was found in the 3.5 x 10 mm and 3.5 x 7 mm stent groups. CONCLUSIONS: The experimental model was thought to simulate adequately the geometry of the human ICA and, thus, would be an effective tool for the research and testing of neurovascular devices. The length of the stent is 1 factor influencing the navigability in tortuous vessels.

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.

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 of SPA bone densitometry for heel bone].

The design principles of a bone densitometer for heel bone based on SPA are introduced here, which include the principles of mechanical system and control system. The main technical index and advantages of the device have also been discussed in the paper.

Predominance of HIV-1 subtype A and D infections in Uganda.

To better characterize the virus isolates associated with the HIV-1 epidemic in Uganda, 100 specimens from HIV-1-infected persons were randomly selected from each of two periods from late 1994 to late 1997. The 200 specimens were classified into HIV-1 subtypes by sequence- based phylogenetic analysis of the envelope (env) gp41 region; 98 (49%) were classified as env subtype A, 96 (48%) as D, 5 (2.5%) as C, and 1 was not classified as a known env subtype. Demographic characteristics of persons infected with the two principal HIV-1 subtypes, A and D, were very similar, and the proportion of either subtype did not differ significantly between early and later periods. Our systematic characterization of the HIV-1 epidemic in Uganda over an almost 3-year period documented that the distribution and degree of genetic diversity of the HIV subtypes A and D are very similar and did not change appreciably over that time.

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.

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.