Analysis of mother-to-infant transmission of hepatitis C virus: Quasispecies nature and buoyant densities of maternal virus populations

Mother-to-infant transmission of hepatitis C virus (HCV) was analyzed by sequencing of viral RNA and semiquantitative polymerase chain reaction following ultracentrifugation of maternal sera. In two mother-infant pairs, the hypervariable region 1 (HVR1) and carboxyl terminus of envelope 1 (E1) were sequenced. Both viral sequences in the infants were less diverse than those of their mothers. Although the E1 sequences were almost identical in each mother-infant pair, the HVR1 sequences of the infants were related, but not identical, to those of the mothers. Serial examinations of one infant revealed that the HVR1 nucleotide sequence did not change from 10 days to 3 months of age. In six mothers with uninfected infants, all of the dense fractions of sera contained significant amounts of HCV RNA, whereas in six mothers with infected infants, only two of those fractions contained significant amounts of HCV RNA. These results indicate that the strains of HCV detected in the infants were not dominant in the mothers, but were still transmissible to the infants. As dense fractions are known to contain antibody-bound HCV particles, maternal antibodies against HCV may inhibit viral transmission. J. Med. Virol. 51:225–230, 1997. © 1997 Wiley-Liss, Inc.     

Differential flotation centrifugation study of hepatitis C virus and response to interferon therapy

Hepatitis C virus (HCV) appears to circulate in various forms such as native virion, immune complexes, and nucleocapsids during chronic infections. To determine the association of the physicochemical properties of HCV and its response to interferon therapy in patients with chronic hepatitis C, we examined pretreatment serum samples from 43 patients with HCV RNA who had received interferon therapy, using differential flotation centrifugation in a NaCl solution with a density of 1.063 g/ml. After centrifugation, the ratio of HCV RNA in the top and bottom fractions was determined by the polymerase chain reaction and expressed as T/B. Patients with a sustained response to IFN therapy were found to have higher T/B ratios than transient responders who relapsed after treatment (P < 0.01) and nonresponders (P < 0.01). With regards to HCV genotypes, patients with genotype 1b had higher T/B ratios in the sustained response group than in the nonsustained response group (P = 0.001), but patients with genotype 2 had a similar distribution of T/B among the 3 response groups (not significant). These findings indicate that the physicochemical properties of HCV affect the effectiveness of interferon therapy, particularly in patients with genotype 1b. J. Med. Virol. 52:190–194, 1997. © 1997 Wiley-Liss, Inc.     

Comparison of different urine culture methods in urinary tract infection

BackgroundMidstream urine specimen cultures play an important role in assisting clinicians to choose antibiotics to remove urinary tract pathogens. At present, there are many culture methods for obtaining midstream urine specimens. In this article, different urine culture methods were compared to improve the detection rate of pathogenic bacteria in patients with urinary tract infections (UTIs).MethodsUrina sanguinis specimens were collected from 90 outpatients and inpatients who had UTI symptoms, had been clinically diagnosed with UTI between February and March 2021, and had been tested within 2 h using the conventional 1-µL culture method, the modified 100-µL culture method, and the centrifugal urine sediment culture method. After incubation at 35 °C with 5% carbon dioxide (CO₂) for 48 h, the detection rates of pathogenic bacteria in UTIs were compared among the three methods.ResultsA total of 75 strains of pathogenic bacteria were detected in the 90 specimens. The positive detection rates of pathogenic microorganisms were 23.33%, 73.33%, and 75.56% for the conventional 1-µL culture method, the modified 100-µL culture method, and the centrifugal urine sediment culture method, respectively. Among the patients who used antibiotics before the collection of the urine specimens, the positive detection rates of pathogenic microorganism were 16.00%, 66.00% and 66.00% for the conventional 1-µL culture method, the modified 100-µL culture method, and the centrifugal urine sediment culture method, respectively. In the specimens grown aseptically using the conventional 1-µL culture method, a similar average number of colonies was found using the modified 100-µL culture method and the centrifugal urine sediment culture method. Among the specimens with bacterial growth in the conventional 1-µL culture method, the average colony numbers of the three methods were similar.ConclusionsThe modified 100-µL culture method and the centrifugal urine sediment culture method greatly improved the positive detection rates of pathogenic bacteria in patients with UTIs.     

Density differences between spermatozoa with antisperm autoantibodies and spermatozoa covered with antisperm antibodies from serum.

Autoantibody-carrying spermatozoa from infertile men were processed using a discontinuous Percoll gradient. The treatment yielded a sperm fraction with significantly reduced antibody loading on the sperm head but did not affect antibody binding to the sperm tail. The immunodepleted sperm preparation demonstrated fertilizing ability when used in in-vitro fertilization. Normal, antibody-free semen samples were coated in vitro with antisperm antibodies from serum, followed by Percoll gradient centrifugation. No significant separation of antibody-free spermatozoa was obtained, regardless of the antibody binding site.     

Bacterial contamination and sperm recovery after semen preparation by density gradient centrifugation using silane-coated silica particles at different g forces

The effects of density gradient centrifugation through silane-coated silica particles (PureSperm) using 100, 200, 300 and 500 g on bacterial contamination of sperm samples and recovery of motile spermatozoa from sperm samples were investigated with conventional culturing techniques and microscopic visual assessment. The recovery of motile spermatozoa was variable and was not improved using 500 g compared to the recommended 300 g. The bacterial contamination was highly decreased by gradient centrifugation through PureSperm and was almost abolished when strict aseptic techniques were used, with changes to sterile Pasteur pipettes and tubes prior to washing procedures.     

微柱离心-药脂比测定脂质体药物包封率

目的建立一种准确测定脂质体包封率的方法。方法以氟吡洛芬、酮洛芬、拓扑替康为模型药物脂质体,通过葡聚糖凝胶(Sephadex G-50)微柱离心去除游离药物,以HPLC法测定药物含量,以定磷法测定磷脂含量,通过药脂比测定药物包封率。结果无需将脂质体完全洗脱,通过检测洗脱脂质体中的药脂比能够准确测定脂质体包封率;对于水溶性药物拓扑替康,若将脂质体完全洗脱,则可能无法将游离药物完全分离。结论在一定条件下微柱离心去除游离药物,通过药脂比测定包封率方便准确。     

Improved techniques for collecting motile spermatozoa from human semen

A simple apparatus to collect moving sperm by non-traumatic means which can be used for artificial insemination is described. The technique is based on enhancing the process of migration from the seminal fluid into a top-layered artificial medium in an ordinary test tube. This has been achieved by controlling 3 main variables: 1) The dilution of migrated sperm was minimize by using only 0.5 ml of the medium layered onto 1 ml semen; 2) increasing the surface area between these media by turning the test tube from a vertical to almost a horizontal position; 3) stimulating sperm activity by incubation at 37 degrees C under air: 5% CO2 for 30 min. When restored to a vertical position approximately 0.3 ml medium, sufficient for most AIH or IVF procedures, was gently aspirated. The effects of these variables on the rate of sperm migration was tested one at a time, and increments that ranged from 20% to as much as 10-fold were detected. When these 3 variables were optimized and 58 semen specimens analyzed, it was found that motility increased from 42 to 87%, velocity from 24.5 to 27.3 micron per sec, whilst abnormal forms dropped from 37 to 15%. The final concentration of motile sperm was 23 X 10(6)/ml compared to an original mean concentration of 34 X 10(6)/ml, indicating a relative recovery of 68%. Oligoasthenospermic specimens revealed similar changes in sperm motility, velocity, morphology and recovery. However, due to the low initial content of moving sperm (4.8 X 10(6)/ml), their final concentration was also low (2.7 X 10(6)/ml). Such specimens required additional preliminary preparation to increase the sperm concentration prior to the migratory procedure.     

Theoretical requirements for the density separation of platelets with comparison of continuous and discontinuous gradients

In continuous density gradients platelets will approach their equilibrium position with exponential deceleration. The time taken to reach an acceptable approximation to equilibrium will depend upon platelet radius, density and medium viscosity. If centrifugation is stopped before equilibrium has been achieved then platelet “subpopulations” produced will not be density dependent. Discontinuous gradients take longer to reach equilibrium. Separation of platelets by density must conform to theoretical physical criteria.     

Mononuclear cell (MNC) collection by continuous-flow centrifugation (CFC)

Mononuclear cell separation and collection by continuous-flow centrifugation relates to centrifugal acceleration, total blood processed, peripheral blood concentration, and probable mobilization from extravascular sites during the procedure. At low-G forces, 70–80% of mononuclear cells are cleared. As G forces are increased, 95–100% of mononuclear cells can be cleared, but excessive platelet depletion occurs. An average of three billion cells were harvested from 2–3 liters of blood, rising to nine billion when 6 liters were processed. The total blood processed is defined by citrate infusion rates and ionized calcium changes. Large quantities of mononuclear cells were collected from normal donors without any significant depletion. If the objective of harvesting mononuclear cells is depletion, intensive schedules may be needed.