Inquiries on intraspousal transmission of hepatitis C virus: benefits and limitations of genome sequencing and phylogenetic analysis.

During recent years, courts had to decide in some notable cases whether or not defendants infected the plaintiffs with HIV. Lawsuits on the transmission of other viral pathogens up to now have hardly become known. We report here on the use of genome sequencing and phylogenetic analysis to investigate possible intraspousal transmission of hepatitis C virus (HCV). The high degree of genetic relatedness observed in phylogenetic analysis among the HCV strains isolated from the couple demonstrates that the man and the woman are infected with the same isolate of the virus. After other plausible routes of infection have been virtually excluded by anamnestic and conventional epidemiological evaluation we could infer that the man most probably has infected his girlfriend with HCV. This conclusion was further supported by the finding that both are also infected with closely related isolates of GB virus C (GBV-C). Thus, the results from molecular biological investigations and epidemiological evaluation are complementary pieces of evidence in inquiries on possible intraspousal transmission of HCV.     

Over-the-counter vaginal contraceptive and spermicide drug products containing nonoxynol 9; required labeling. Final rule

The Food and Drug Administration (FDA) is issuing a final rule establishing new warning statements and other labeling information for all over-the-counter (OTC) vaginal contraceptive drug products (also known as spermicides, hereinafter referred to as vaginal contraceptives or vaginal contraceptives/spermicides) containing nonoxynol 9 (N9). These warning statements will advise consumers that vaginal contraceptives/spermicides containing N9 do not protect against infection from the human immunodeficiency virus (HIV), the virus that causes acquired immunodeficiency syndrome (AIDS), or against getting other sexually transmitted diseases (STDs). The warnings and labeling information will also advise consumers that use of vaginal contraceptives and spermicides containing N9 can irritate the vagina and rectum and may increase the risk of getting the AIDS virus (HIV) from an infected partner. This final rule is part of FDA's ongoing review of OTC drug products. FDA is issuing this final rule after considering public comments on its proposed regulation, and all relevant data and information on N9 that have come to our attention.     

New alleles and mutational events in D12S391 and D8S1132: sequence data from an eastern German population.

To investigate the DNA mutation rate and pattern in the hypervariable short tandem repeat (STR) locus D12S391 and in the locus D8S1132, samples from an eastern German population (Dresden area) were analysed. A duplex PCR was applied, using short amplification products for D12S391 (129-177bp) and a modified reverse primer for D8S1132 (127-182bp). The sequences of some rare and new variant alleles are described. At the locus D12S391, 13 regular and six incomplete alleles with different lengths were found, exhibiting several sequence structures. Two isolated father/child mismatches were observed in a total of 648 meioses. Novel alleles 13.1, 14.1 and 27 were discovered at the locus D8S1132. Three parent/child mismatches were found in a total of 672 meioses.     

Effect of murine retroviral infection on hair and serum levels of cocaine and morphine.

LP-BM5 retrovirally infected female C57BL/6J mice were administered cocaine, morphine or both by daily intraperitoneal injection for 9 weeks. Drug concentrations were measured by radioimmunoassay in serum and in hair extracts. Hair samples obtained from all drug-treated mice were positive for the drug injected, while none of the saline-treated mice had detectable drug levels in hair or serum. The average morphine concentration obtained from non-infected mice was 11 ng/mg hair whereas the amount found in the LP-BM5-infected mice was significantly higher (20 ng/mg hair). Mice injected with both morphine and cocaine were given 50% of the regular dose of each drug and drug levels in the hair of these animals were approximately half that of mice injected with the full dose of the single drug. Non-infected mice treated with both drugs had a mean value of 7 ng morphine/mg hair and 374 ng cocaine/mg hair while retrovirus-infected mice had significantly higher concentrations, 10 ng morphine/mg hair and 1160 ng cocaine/mg hair (P less than 0.001). Serum concentrations of cocaine and morphine were significantly higher (P less than 0.01) in the retrovirus-infected animals from 40 min to 1.5 h. The increased concentrations of cocaine and morphine in serum during retrovirus infection are accompanied by a significant increase in the amount of drug incorporated into the hair matrix. This change indicates that retroviral infection may influence the disposition of these drugs in the systemic circulation.     

Identification and synthesis of some contaminants present in 4-methoxyamphetamine (PMA) prepared by the Leuckart method

The clandestine synthesis of ring and side chain modified phenylisopropylamines continues to be a major source of these drugs of abuse. One method used for the synthesis of the amphetamine and related compounds involves the treatment of the appropriate ketone with formamide or ammonium formate followed by acid hydrolysis of intermediate N-formyl derivative. In this paper the synthesis of 4-methoxyamphetamine (PMA, 1) by the Leuckart method is investigated. The identification by means of gas chromatography-mass spectrometry (GC-MS) of methoxy derivative of N-(beta-phenylisopropyl)benzaldimine 9, methoxy derivative of N-(beta-phenylisopropyl)benzyl methyl ketimine 5, 1-(4-methoxyphenyl)-N-(4-methoxybenzyl)-2-propanamine 10, (RR/SS) and (RS) 1-(4-methoxyphenyl)-N-[2-(4-methoxyphenyl)-1-methylethyl]-2-propanamine 6a-6c, (RR/SS) and (RS)-1-(4-methoxyphenyl)-N-methyl-N-[2-(4-methoxyphenyl)-1-methylethyl]-2-propanamine 7a-7c, (RR/SS) and (RS)-1-(4-methoxyphenyl)-N-formyl-N-[2-(4-methoxyphenyl)-1-methylethyl]-2-propanamine 8a-8c in crude PMA, are reported. The identity of these compounds was confirmed by independent synthesis of reference compounds. The NMR, MS, IR data, stereochemistry and some chromatographic properties of synthesized compounds are discussed. Finally, the results of the GC-MS analysis of illicitly prepared tablets, containing PMA 1 and 4-methoxymethamphetamine (PMMA, 2), are outlined. The presence of 4-methoxydimethylamphetamine 11, 4-methoxyethylamphetamine 12, and 4-hydroxymethamphetamine 13 are reported in these tablets. The identity of 2, 11, and 12 was confirmed by their independent synthesis.     

抗人转铁蛋白单链抗体的研制及部分性能研究

研制人转铁蛋白的单链抗体。以纯化人转铁蛋白免疫小鼠,制备小鼠脾脏 mRNA,RT-PCR 扩增重轻链可变区基因;Linker 连接及引入 Sfi Ⅰ和 NotⅠ酶切位点构建单链抗体基因;同载体 pCANTAB 5E 连接,转化 E.coliTG1细胞,以 M13K07挽救制备噬菌体抗体呈现文库,筛选抗原阳性重组噬菌体粒子(Panning);感染 E.coliHB2151菌株,制备可溶抗体并检测其性质;测定抗体基因序列并分析同源性及结构特征。制备出4株分泌抗人转铁蛋白可溶抗体的 E.coli HB2151菌株。抗体基因只由小鼠抗体的轻重链可变区基因构成,开放读码,无终止子,并紧接一末尾为一琥珀终止密码的 E 末端序列,其对应氨基酸序列 V_H 和 V_L 部分均遵从抗体可变区特征。实验证明,可把基因工程抗体技术应用于法医血清学,以制备新型抗体试剂。     

Degradation pathways of 4-methylmethcathinone in alkaline solution and stability of methcathinone analogs in various pH solutions

Analogs of methcathinone (MC), a psychoactive stimulant, are in circulation all over the world. These analogs have been assumed to be unstable in alkaline solutions, as is MC itself. The aims of this study were: (i) to identify the degradation products of 4-methylmethcathinone (4-MMC), a typical MC analog, in solution at pH 12 and to determine the degradation pathway, (ii) to investigate the effects of antioxidants such as l-ascorbic acid and sodium sulfite on the degradation of 4-MMC, and (iii) to investigate the stability of seven MC analogs (4-MMC, 4-, 3-, or 2-fluoromethcathinone, 4-methoxymethcathinone, N-ethylcathinone, and N,N-dimethylcathinone) in solutions at different pHs.1-(4-Methylphenyl)-1,2-propanedione (MPPD), 4-methylbenzoic acid (MBA), N,4-dimethylbenzamide (DMBA), and N-acetyl-4-MMC (N-Ac-4-MMC) were identified as the degradation products of 4-MMC in pH 12 solution by gas chromatography-mass spectrometry. There are two degradation pathways for 4-MMC as follows: (a) 4-MMC→MPPD→MBA→DMBA and (b) 4-MMC→N-Ac-4-MMC. Oxidants such as dissolved oxygen were presumed to be involved in this degradation based on the suppressive effects generated by the addition of antioxidants. All of the seven MC analogs tested were stable in acidic (pH 4) solution but degraded in neutral-to-basic solutions. Their degradation rates increased with increasing pH, and varied with their chemical structures. These findings will be very useful for not only forensic analysis but also future pharmacokinetic analysis.     

Quantification of mitochondrial DNA in human blood cells using an automated detection system

The 4977 bp deletion of mitochondrial DNA (mtDNA) accumulates in postmitotic tissues with advancing age. The purpose of our study was to detect and quantify these deletion even in blood cells with a high turnover activity. Whole venous blood, isolated human platelets and peripheral blood mononuclear cells (PBMCs) were collected from 10 unrelated donors aged 20-71 years and total DNA was extracted. PCR was performed for total and mutated mtDNA using two different primer pairs and two fluorogenic probes labeled with the fluorescent dyes FAM and VIC. Specific PCR products were generated, detected and quantified in a real-time PCR. The amplification products of total and deleted mtDNA could be detected in each sample and did not exhibit any differences in the amount of the deleted mtDNA in whole blood, human platelets or PBMCs. Our data did not show any accumulation of the 4977 bp deletion with increasing age as it was observed for several other tissues.     

Identification and phylogenetic analysis of hepatitis C virus in forensic blood samples obtained from injecting drug users

Injecting drug users (IDUs) are a high-risk group for contracting hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) infections. In Japan, data on the prevalence of those blood-borne viruses among IDUs are very limited. Blood samples were obtained from 12 cadavers of IDUs sent to Nagoya City University for the purpose of judicious autopsy and two alive IDUs with hepatitis C referred to a local hospital at the same period. The viruses were detected by polymerase chain reaction and phylogenetic analysis was performed. Two (16.6%) of the 12 autopsy cases were positive for HCV, but no case was positive for either HBV or HIV. Phylogenetic analysis of the two HCV isolates revealed that one was classified into genotype 1b and another was genotype 2b. Furthermore, nucleotide sequences of two isolates recovered from IDUs with hepatitis C were identical, that indicated the transmission of HCV between them, and those HCV were phylogenetically classified into genotype 2a. The prevalence of HCV infection among IDUs in Japan, despite the case of judicious autopsy, seems to be high, but HIV infection seems to be rare. The transmission of HCV between IDUs was demonstrated, and this indicates that phylogenetic analysis would applicable to also forensic analysis. HCV isolates identified in this study did not phylogenetically segregate, thus multiple transmission route of HCV among IDUs seems be exist in Japan.     

The different mobility of complementary strands depends on the proportion AC/GT

The electrophoretic mobility of DNA fragments on denaturing polyacrylamide gel depends on various factors. One of these is the base composition of a single-stranded DNA (ssDNA). We confirmed that one strand and its complementary strand of polymerase chain reaction (PCR) products migrated with different mobilities in all alleles detected at 12 out of the 13 short tandem repeat (STR) loci studied. The mobility differences between complementary strands (MD) were also observed regardless of end-polishing with Pfu DNA polymerase. MD was therefore not influenced by additional nucleotides to each strand of the PCR products. We then analyzed the relation between MD and the base composition using one representative allele at each of the 13 loci. The results indicated that MD was affected by the adenine plus cytosine (AC) content in the ssDNA and was proportional to the values of the AC content divided by the guanine plus thymine (GT) content in the AC-rich strand (the proportion AC/GT). When the proportion AC/GT was well-balanced, MD decreased. The same tendency was observed even in the end-polished strands. In this study, the electrophoretic mobility of an ssDNA on denaturing polyacrylamide gels was shown to depend on the proportion AC/GT. Unless the same side of the PCR products is labelled in the context of a PCR-based STR typing, distinct alleles may be mistaken for identical ones because of the different mobility of complementary strands. Accordingly, the labelled strand should be described if only one strand of the PCR products is detected. When using an allelic ladder marker as a size standard, the labelled side should be unified between STR alleles and the allelic ladder alleles.     

Rat bite fever, a fatal case of Streptobacillus moniliformis infection in a 14-month-old boy

Streptobacillus moniliformis is the primary cause of rat bite fever in North America. Children under 12 years of age are primarily infected, presenting with an acute syndrome of fever, rash, and polyarthritis. Common vectors include rats and mice. Transmission is predominantly from a bite or scratch, but contact with or ingestion of food contaminated with feces or saliva has also been reported. We report a fatal case of S. moniliformis infection in a 14-month-old-boy. At home, the child was exposed to filthy living conditions and the family had pet ferrets. Autopsy revealed a red-pink, mostly confluent rash and a circumscribed area suspicious for a possible bite mark. Cerebrospinal fluid cultures were positive for S. moniliformis. This case highlighted key features, such as the morbiliform rash, but lacked lymphadenopathy or joint manifestations. It is important to consider rat or rodent exposure as a source of infection.     

The detection of acetylcodeine and 6-acetylmorphine in opiate positive urines

Acetylcodeine (AC), an impurity of illicit heroin synthesis, was investigated as a urinary biomarker for detection of illicit heroin use. One hundred criminal justice urine specimens that had been confirmed positive by GC/MS for morphine at concentrations >5000 ng/ml were analyzed for AC, 6-acetylmorphine (6AM), codeine, norcodeine and morphine. The GC/MS analysis was performed by solid phase extraction and derivatization with propionic anhydride. Total codeine and morphine concentrations were determined by acid hydrolysis and liquid/liquid extraction. AC was detected in 37 samples at concentrations ranging from 2 to 290 ng/ml (median, 11 ng/ml). 6AM was also present in these samples at concentrations ranging from 49 to 12 600 ng/ml (median, 740 ng/ml). Of the 63 specimens negative for AC, 36 were positive for 6AM at concentrations ranging from 12 to 4600 ng/ml (median, 124 ng/ml). When detected, the AC concentrations were an average of 2.2% (0.25 to 10.2%) of the 6AM concentrations. There was a positive relationship between AC concentrations and 6AM concentrations (r=0.878). Due to its very low concentration in urine, AC was found to be a much less reliable biomarker for illicit heroin use than 6AM in workplace or criminal justice urine screening programs. However, AC detection could play an important role in determining if addicts in heroin maintenance programs are supplementing their supervised diacetylmorphine doses with illicit heroin.     

Genotypic Polymorphisms of Hepatitis B Virus Provide Useful Information for Estimating Geographical Origin or Place of Long‐term Residence of Unidentified Cadavers

Increasing numbers of unidentified cadavers are a major problem. We have developed a new method for providing identification information that can determine the geographical origin or place of long‐term residence of unidentified cadavers based on genotypic polymorphisms of hepatitis B virus (HBV) known to correlate with their geographical distribution. PCR of serum samples detected HBV DNA from 4 (3.9%) of 102 randomly selected Japanese forensic cadavers. Multiplex PCR did not detect multiple HBV genotypes from any single cadaver, confirming the absence of coinfection. Phylogenetic tree analysis based on a 485‐bp mutant region of the HBV S gene successfully classified the HBV genotypes into A to J. Among 10 HBV‐infected cadavers, 8 had genotype Ce/C2, a genotype prevalent in East Asia, and 2 had genotype Bj/B1, a Japanese‐specific genotype. HBV genotypic polymorphisms correlate with the geographical distribution of the virus and thus provide important information for identifying unidentified cadavers infected with HBV.     

Regioselective synthesis of isotopically labeled Δ9-tetrahydrocannabinolic acid A (THCA-A-D(3)) by reaction of Δ9-tetrahydrocannabinol-D(3) with magnesium methyl carbonate

For the reliable quantification of Δ9-tetrahydrocannabinolic acid A (THCA-A), the biogenetic precursor of Δ9-tetrahydrocannabinol (THC), in biological matrices by LC-MS/MS and GC-MS(/MS), an isotopically labeled internal standard was synthesized starting from Δ9-tetrahydrocannabinol-D(3) (THC-D(3)). Synthesis strategy was based on a method reported by Mechoulam et al. in 1969 using magnesium methyl carbonate (MMC) as carboxylation reagent for the synthesis of cannabinoid acids. Preliminary experiments with THC to optimize yield of the product (THCA-A) resulted in the synthesis of the positional isomer tetrahydrocannabinolic acid B (THCA-B) as a byproduct. Using the optimized conditions for the desired isomer, THCA-A-D(3) was prepared and isolated with a yield of approx. 10% after two synthesis cycles. Isotope purity was estimated to be >99% by relative abundance of the molecular ions. The synthesized compound proved to be suitable as an internal standard for quantification of THCA-A in serum and hair samples of cannabis consumers.     

Postmortem Detection of Hepatitis B,C, and Human Immunodeficiency Virus Genomes in Blood Samples from Drug‐Related Deaths in Denmark*

Abstract: Blood‐borne viral infections are widespread among injecting drug users; however, it is difficult to include these patients in serological surveys. Therefore, we developed a national surveillance program based on postmortem testing of persons whose deaths were drug related. Blood collected at autopsy was tested for anti‐HBc, anti‐HBs, anti‐hepatits C virus (HCV), or anti‐human immunodeficiency virus (HIV) antibodies using commercial kits. Subsets of seropositive samples were screened for viral genomes using sensitive in‐house and commercial polymerase chain reaction (PCR) assays. Hepatitis B virus (HBV) DNA was detected in 20% (3/15) of anti‐HBc‐positive/anti‐HBs‐negative samples, HCV RNA was found in 64% (16/25) of anti‐HCV‐positive samples, and HIV RNA was detected in 40% (6/15) of anti‐HIV‐positive samples. The postmortem and antemortem prevalences of HBV DNA and HCV RNA were similar. Postmortem HIV RNA testing was less sensitive than antemortem testing. Thus, postmortem PCR analysis for HBV and HBC infection is feasible and relevant for demonstrating ongoing infections at death or for transmission analysis during outbreaks.     

Cytomegalic inclusion disease of the salivary glands in sudden infant death syndrome

The parotid gland and/or the submandibular gland in SIDS cases (180 from Berlin und 75 cases from Hamburg) were examined by means of HE staining, immunohistochemical analysis, in situ hybridization and electron microscopy. The SIDS cases were taken from the last 10 years; the age of the children ranged from 2 weeks to 1 year. Typical cytomegaly inclusion bodies were recognized in 10% (18 cases from Berlin; more girls than boys) and 7% (6 cases from Hamburg; more boys than girls). Our files indicate that the frequency of CMV infection was not age-dependent within the first 12 months of life. Using immunohistochemical analysis and in situ hybridization, virus substances were detected in cytomegal cells as well as in morphologically uninfected cells. The literature on the clinical and epidemiological aspects of cytomegaly indicates that a localized CMV infection of the salivary glands does not sufficiently explain the sudden death of these infants; however, it should be emphasized that cytomegaly can influence the immunological status of the organism.     

Post-mortem analysis of formic acid disposition in acute methanol intoxication

Fifteen cases of fatal massive methanol intoxication have been investigated. Victims received either no treatment or ethanol therapeutic treatment. Methanol poisoning cases were classified in three groups according to survival time: more than 3 days (group 1), up to 3 days (group 2) and few hours (group 3). Body distribution of methanol and formic acid, as the main metabolite, was analyzed in blood and in different organs (brain, kidney, lung and liver). Relationships between formic acid concentration in the different tissues, survival time and type of treatment applied to victims were studied. Formic acid in blood and tissues was analyzed by head space gas chromatography (head space-GC) with FID detector, previous transformation in methyl formate, essentially as described by Abolin. Formic acid concentration was between 0.03 and 1.10g/l in the samples under study. A good correlation between blood and brain, but poor between blood and the remaining tissues was found. Obtained data suggested that the use of blood and brain could help to improve the analysis of formic acid intoxication. The best correlation among organs was found between lung and kidney for all groups (r(2)=0.91, 0.84 and 0.87, corresponding to groups 1, 2 and 3, respectively). Lethality index was defined as LI = (concentration of formic acid in blood in (g/l)/0.5) x 100, taking into account that 0.5g/l is the concentration reported by Mahieu in severe methanol poisoning. LI parameter was used to estimate formic acid incidence on the lethality of methanol poisoning cases. LI showed a good correlation with total formic acid concentration of the different tissues analyzed (r(2)=0.80). Furthermore, LI allowed us to discriminate between individuals that received therapeutic treatment and survived different periods. LI>100 indicated a severe intoxication and short survival time if the victim was assisted with ethanol therapy and hemodialysis was not applied. With regard to victims who received no therapeutic treatment and died in few hours, LI was in the range 40-100. LI was below 40 for individuals that survived more than 3 days and hemodialysis was not performed. Results showed the importance of performing formic acid analysis to diagnose severe methanol intoxication in post-mortem cases.     

不同温度下血样中毒鼠强的稳定性

目的探讨不同保存温度下血样中毒鼠强的稳定性。方法配制毒鼠强质量浓度为0.5μg/mL的血液样品,分别置于45℃、25℃和4℃环境中保存,在配制当天及存放3、12、18和39d用气相色谱-质谱联用法测定其质量浓度,运用SPSS统计软件对结果进行统计分析。结果45℃、25℃下血样存放3d毒鼠强质量浓度基本不变,存放4～12d毒鼠强质量浓度有明显下降,存放12d后毒鼠强质量浓度缓慢下降;4℃下血样存放12d毒鼠强质量浓度基本不变,存放13～18d毒鼠强质量浓度有明显下降,存放18d后毒鼠强样品质量浓度缓慢下降。结论血样中毒鼠强质量浓度在最初的3d内稳定,此后浓度下降。保存温度对血样中毒鼠强质量浓度有影响。     

Histopathological findings in 851 autopsies of drug addicts, with toxicologic and virologic correlations

This investigation was carried out on 851 consecutive judicial autopsies of drug addicts who died mostly of heroin overdose from 1977 to 1996. Research of anti-HIV/HBV/HCV antibodies was performed, and histologic sections were retrospectively reviewed. More than 65% were HBV/HCV-positive and about 17% HIV-positive; females were HIV-positive more often than males. Intracranial microhemorrhages were frequently found; cerebral infectious diseases were rare. Inflammatory heart lesions, myocardial fibrosis, and acute ischemia were common. Interstitial nephritis (found in about 8%) was more frequent in females, in older patients, and in those carrying HIV infection; glomerular sclerosis was detected in about 12%. Acute bronchitis and/or pneumonia was demonstrated in 12%, without significant association with HIV infection; pulmonary hemorrhages, foreign body granulomas, and food aspiration were also commonly seen; hyperplasia of pulmonary perivascular lymphatic tissue was rather characteristic. Liver was carrying steatosis in 66.3% and/or hepatitis in 64.5%; acute hepatitis was more frequent in females, chronic hepatitis in older subjects and in those proven positive for hepatotropic viruses; cirrhosis occurred more often in older patients, in those carrying virus infection, and in consumers of nonnarcotics drugs such as ethanol. No pathologic finding was clearly related to drug abuse; therefore, during autopsy, drug addiction can be suspected, but anamnestic and circumstantial data are needed to lead pathologists to request toxicologic analysis to ascertain the cause of death. The present investigation emphasizes that, in addition to the risk of death by overdose, the high incidence of acute and chronic diseases could seriously undermine the health status of heroin and/or other drug consumers.     

人脑挫伤后GFAP,PCNA的法医病理学研究

采用在实际工作中所遇到的脑挫伤案例标本 ,利用免疫组织化学染色 ,观察人脑挫伤后GFAP ,PCNA的改变 ,用图象分析仪对免疫组织化学染色阳性细胞的面积和灰度等进行定量测量 ,EpiInfo统计软件分析 ,得出人脑挫伤后GFAP ,PCNA变化的时间性规律 ,为脑挫伤形成时间推断 ,提供新的形态学依据。GFAP阳性细胞面积、灰度伤后24h即有显著增加 (P<0 01) ,7天达高峰 (P<0 01) ,以后有逐渐下降的趋势 ;PCNA阳性细胞面积、灰度于伤后4天达到最大值 (P<0 01) ,以后逐渐下降。因此 ,伤后人脑组织GFAP、PCNA免疫组织化学染色阳性细胞面积、灰度改变有时间规律 ,GFAP、PCNA可作为2天到20天间不同时间脑损伤的推断指标。