HIV rapid testing in a Ugandan blood bank
Use of PCR to detect chloroquine-resistant Plasmodium falciparum
Link between polymorphisms of the estrogen receptor-beta gene and cardiac disease risk
Role of declining p53 function in the aging process
Plasma annexins in relation to antiphospholipid antibody status
Function of herpes simplex virus glycoproteins
Rapid testing for HIV has become the standard in resource-limited settings for programs addressing voluntary counseling and testing and the prevention of mother-to-child HIV transmission. Diagnosing HIV infection using serology, usually duplicate enzyme immunoassays with confirmation by Western blotting, is neither feasible nor practical in many developing countries due to technical and financial constraints. In Uganda and many other countries, several rapid testing algorithms, often based on the availability of test kits rather than the proven specifications of an algorithm, are being employed. The Uganda Blood Transfusion Service supplies more than 140,000 units of blood per year to hospitals in Uganda. Blood donations are collected at the blood donation center in Kampala and at multiple donation centers nationwide. In addition to the blood bank in Kampala, there are four other major regional blood banks in Uganda—in Gulu in the north, Mbale in the east, Fort Portal in the west, and Mbarara in the south. The headquarters ensures that health sector policy on blood transfusion is implemented and that all blood collection sites adhere to quality assurance and safety requirements. The authors reported on an extensive HIV rapid test validation study conducted among Ugandan blood bank donors at low risk for HIV infection. The operational characteristics of four readily available commercial HIV rapid test kits (Determine HIV-1/2, Uni-Gold Recombigen HIV, OraQuick HIV-1, and HIV-1/2 Stat-Pak) were determined with 940 donor samples and used to select a serial testing algorithm. The tests were chosen based on their ease of use, flexible storage requirements, and large-scale distribution and availability in Uganda. Uni-Gold Recombigen HIV was used as the screening test, followed by HIV-1/2 Stat-Pak for reactive samples. OraQuick HIV-1 testing was performed if the first two test results were discordant. This algorithm was then tested with 5,252 blood donor samples, and the results were compared to those of enzyme immunoassays and Western blotting. The unadjusted algorithm sensitivity and specificity were 98.6 percent and 99.9 percent, respectively. The adjusted sensitivity and specificity were 100 percent and 99.96 percent, respectively. The authors concluded that this HIV testing algorithm is a suitable alternative to enzyme immunoassays and Western blotting for Ugandan blood donors.
Eller LA, Eller MA, Ouma BJ, et al. Large-scale human immunodeficiency virus rapid test evaluation in a low-prevalence Ugandan blood bank population. J Clin Microbiol. 2007;45:3281–3285.
Correspondence: Leigh A. Eller at firstname.lastname@example.org
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Chloroquine became the antimalarial agent of choice during the second half of the 20th century because of its efficacy, affordability, ease of use, and low toxicity. However, the simultaneous appearance of chloroquine-resistant Plasmodium falciparum malaria in the late 1950s in Southeast Asia and South America and its subsequent spread to most regions to which malaria is endemic have dramatically restricted the usefulness of this drug as a therapeutic or chemoprophylactic agent. Chloroquine resistance in falciparum malaria is associated with mutations in the Pfcrt gene, which encodes the P. falciparum chloroquine-resistance transporter (PfCRT), a transmembrane protein in the digestive vacuole of malaria parasites. Chloroquine-resistant Plasmodium falciparum (CRPF) malaria isolates in Southeast Asia and sub-Saharan Africa share the same PfCRT haplotype (CVIET; amino acids 72 to 76). It is believed that CRPF malaria emerged in Southeast Asia and spread to sub-Saharan Africa via the Indian subcontinent. Based on this assumption, the authors hypothesized that CRPF isolates in India should possess the same drug resistance haplotype (PfCRT haplotype CVIET) as P. falciparum isolates in Southeast Asia and Africa and that the prevalence of CRPF may be higher and more widespread in India than thought. To test this postulate, the authors used a standardized real-time PCR assay to assess the prevalence and distribution of the PfCRT haplotypes in P. falciparum isolates (n=406) collected from western, central, and eastern states in India and compared them to isolates from South America (Suriname) and Africa (Uganda) to determine if they fit an Old World (CVIET haplotype) or New World (SVMNT haplotype) pattern of geographic origin. Based on the proportion of isolates possessing the molecular marker K76T, the prevalence of chloroquine resistance was high in all five regions of India studied (91 percent), as well as in Uganda (98 percent) and Suriname (100 percent). All isolates from Suriname contained the chloroquine-resistant SVMNT haplotype typical of South American isolates, and 98 percent of isolates from Uganda possessed the chloroquine-resistant CVIET haplotype characteristic of Southeast Asian/ African strains. However, of 246 P. falciparum isolates from across India that contained the molecular marker for chloroquine resistance, 81 percent contained the SVMNT haplotype. The authors concluded that the prevalence of CRPF malaria was high in geographically dispersed regions of India, and the primary haplotype observed, SVMNT, did not support a presumed geographic spread from contiguous Southeast Asia.
Keen J, Farcas GA, Zhong K, et al. Real-time PCR assay for rapid detection and analysis of PfCRT haplotypes of chloroquine-resistant Plasmodium falciparum isolates from India. J Clin Microbiol. 2007;45:2889–2893.
Correspondence: Kevin C. Kain at email@example.com
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The development of atherothrombotic cardiovascular disease is likely polygenic. The cardiovascular effects of estrogen are mediated through binding to specific estrogen receptors at the cytosolic and nuclear level. Two estrogen receptors have been identified: estrogen receptor-α (ER-α) and estrogen receptor-β (ER-β). Polymorphisms of the ER-α gene (ESR1) recently have received attention as possible contributors to cardiovascular disease risk in men and women, but the relationship between genetic variation of the ER-β gene (ESR2) and cardiovascular disease has not been as well studied. Several lines of evidence support a potential role of ESR2 in cardiovascular disease. ER-β is highly expressed in endothelial and vascular smooth muscle cells and has been associated with coronary plaque. In autopsy studies, ER-β expression positively correlates with increased coronary artery plaque area in women and men. Polymorphisms of ESR2 have been associated with left ventricular mass and left ventricular wall thickness in women but not men. ESR2, located on chromosome 14 q22–24, comprises eight exons. The authors evaluated three polymorphisms in the ESR2 gene, as well as their associated haplotypes, with risk of CVD (defined as myocardial infarction or ischemic stroke) among men in the Physicians’ Health Study (PHS) and women in the Women’s Health Study (WHS) using a nested case-control design. The study involved 296 white women and 566 white men who developed cardiovascular disease, each matched 1:1 to a member of the cohort study who remained free from the disease. Blood samples and cardiovascular risk information were collected at baseline. Women, but not men, who developed cardiovascular disease or myocardial infarction, but not ischemic stroke, were more likely to have the rs1271572 polymorphism variant T allele (P=.05 and .02) and less likely to have the rs1256049 polymorphism variant A allele (P=.003 and .004). No associations were observed for rs4986938. In conditional logistic multivariate regression, the rs1271572 variant was associated with increased odds of cardiovascular disease (odds ratio, 1.49; 95 percent confidence interval [CI], 1.10–2.01) and myocardial infarction (odds ratio, 1.46; 95 percent CI, 0.96–2.23), whereas the rs1256049 variant was associated with decreased odds of cardiovascular disease (odds ratio, 0.37; 95 percent CI, 0.17–0.79) and myocardial infarction (odds ratio, 0.25; 95 percent CI, 0.09–0.73) in women. A common haplotype that included the rs1271572 variant was associated with a seven-fold increased risk of myocardial infarction in women. The authors concluded that two tightly linked polymorphisms of ESR2 were associated with risk of cardiovascular disease, particularly myocardial infarction, in women but not men. Additional studies of ESR2 genetic variation and risk of cardiovascular disease are warranted.
Rexrode KM, Ridker PM, Hegener HH, et al. Polymorphisms and haplotypes of the estrogen receptor-β gene (ESR2) and cardiovascular disease in men and women. Clin Chem. 2007;53:1749–1756.
Correspondence: Kathryn M. Rexrode at firstname.lastname@example.org
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Cancer is largely a disease of the elderly, with most tumors arising in the last quarter of life and their frequency increasing exponentially with time. Mice (two- to three-year mean life spans) frequently acquire tumors after 18 months to two years, whereas dogs (12- to 16-year mean life spans) acquire most tumors after 10 years. Humans (75-year mean life spans) acquire tumors with an increased frequency after 50 to 55 years of age. This increased frequency is thought to be caused by the accumulation of DNA mutations in oncogenes or tumor-suppressor genes in individual cells (somatic tissues) over a lifetime. This hypothesis is supported by ample evidence from human and animal models showing that many tumors contain mutations in crucial tumor-suppressor genes and oncogenes. The tumor-suppressor p53 gene is the most frequently mutated gene in human tumors—more than 50 percent of tumors harbor mutations in the p53 gene or the p53 pathway. The p53 protein can be activated by a variety of stress signals and results in various cellular responses, including apoptosis, cell cycle arrest, or senescence through transcriptional regulation of its target genes. Disruption of normal p53 function is in some circumstances a prerequisite for the development or progression of tumors. By preventing these tumors early in life, the p53 gene plays a role in assuring longevity. For example, p53-null mice succumb to tumors within several months, and heterozygous p53 mutant mice develop tumors over a period of a year or more. LiFraumeni syndrome patients with the heterozygous p53 gene display a 50 percent cancer incidence by the age of 30. It has been reported that the function of DNA repair, regulation of cell proliferation, and immune response decrease with age in animals and humans. These findings together raised the hypothesis: Could the function of p53 protein decline with age, which may then contribute to the enhanced mutation frequency and tumorigenesis in the aging process along with the accumulation of DNA mutations? To explore this hypothesis, the authors investigated the activity of the p53 protein and the efficiency of p53 responses to various stresses in inbred mouse strains as a function of their ages. The efficiency of the p53 response to y-irradiation was found to decline significantly in various tissues of aging mice from several inbred strains, including lower p53 transcriptional activity and p53-dependent apoptosis. This decline resulted from decreased stabilization of the p53 protein after stress. The function of the Ataxia-telangiectasia mutated (ATM) kinase declined significantly with age, which may be responsible for the decline in p53 response to radiation. Declining p53 responses to other stresses were also observed in the cultured splenocytes from aging mice. The time of onset of decreased p53 response correlated with the life span of mice; mice that live longer delay their onset of decreased p53 activity with time. These results suggest an enhanced fixation of mutations in older people because of the declining fidelity of p53-mediated apoptosis or senescence in response to stress. They also suggest a plausible explanation for the correlation between tumorigenesis and the aging process.
Feng Z, Hu W, Teresky AK, et al. Declining p53 function in the aging process: a possible mechanism for the increased tumor incidence in older populations. Proc Natl Acad Sci USA. 2007; 104:16633–16638.
Correspondence: Arnold J. Levine at email@example.com
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Recurrent miscarriage affects one percent of all pregnancies. The etiology of such miscarriage is multifactorial, but the idea of an association between recurrent miscarriage (RM) and hereditary and acquired thrombophilias has gained support in recent years. The presence of antiphospholipid (aPL) antibodies is one of the most common causes of acquired thrombophilia and is known to increase the risk of arterial and venous thromboses and adverse pregnancy outcome. While the exact causative mechanism behind the thrombotic events related to the presence of aPL antibodies is still obscure, these antibodies have been shown to have many antigenic targets, such as prothrombin, protein C, protein S, tissue plasminogen activator, and annexins, explaining their thrombophilic potential leading to placental thrombosis and infarctions. Annexins are a family of structurally related proteins that bind to anionic phospholipids in a calcium ion-dependent manner. The best known annexin, annexin V, has anticoagulant properties and the capacity to displace coagulation factors from anionic phospholipid surfaces. Annexin V expression has been observed on the apical surface of syncytiotrophoblasts (STBs), whereas annexin IV expression has been observed in the basal layer of STBs in the placenta. Therefore, annexins, as local anticoagulants in the placenta, could play an important role in maintaining pregnancy, especially during the first trimester. The authors conducted a study to define plasma concentrations of circulating annexins IV and V at the beginning of pregnancy among women with a history of recurrent miscarriage, regardless of their aPL antibody status. The study included 68 women with recurrent miscarriage and 25 controls without a history of adverse pregnancy outcome. The authors determined concentrations of annexins IV and V in plasma using a sandwich enzyme-linked immunosorbent assay technique. Hereditary or acquired thrombophilic disorders were found in 53 percent (36/68) of the patients who experienced recurrent miscarriage. Plasma levels of annexin V were significantly higher at the beginning of pregnancy (P=.03) and at the sixth (P=.01) and eighth (P=.01) weeks of pregnancy in women with aPL antibodies compared with those without aPL antibodies. A tendency towards higher plasma levels of annexin V was observed in those whose pregnancies ended in miscarriage compared to those with successful pregnancy, although the results did not reach statistical significance (P=.10). Plasma levels of annexin IV at the first visit in women with aPL antibodies were similar to those at six and eight weeks of gestation. There were no significant differences in plasma annexin IV levels between women with and without aPL antibodies. The authors concluded that patients with recurrent miscarriage show elevated plasma levels of annexin V in the presence of aPL antibodies. These antibodies could displace annexin from anionic phospholipid surfaces of syncytiotrophoblasts and thereby promote coagulation activation.
Ulander V-M, Stefanovic V, Masuda J, et al. Plasma levels of annexins IV and V in relation to antiphospholipid antibody status in women with a history of recurrent miscarriage. Thrombosis Res. 2007;120:865–870.
Correspondence: Risto Kaaja at firstname.lastname@example.org
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The nuclear envelope of cells is composed of an inner and outer nuclear membrane separated by perinuclear space and connected by nuclear pore complexes. A dense network of lamins forms a rigid girdle underneath the inner nuclear membrane, organizing nuclear pores and chromatin. For viruses that replicate their genomes in the nucleus, the nuclear envelope often serves to confine and orchestrate nucleic acid synthesis and assembly of virus particles. However, the nuclear envelope can also present a formidable barrier to virus egress from cells. Nonenveloped viruses, such as SV 40 and adenoviruses, rupture the nuclear envelope and plasma membrane to gain release from cells. Herpes viruses assemble large capsids in the nucleus but, because they are enveloped viruses, can cross membranes by budding and fusion mechanisms. The authors investigated the nuclear egress of herpes simplex virus (HSV) by characterizing a panel of double mutants lacking pairs of HSV glycoproteins. They showed that an HSV mutant lacking the two putative fusion glycoproteins, gB and gH, failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or just gH showed only minor or no defects in nuclear egress. The authors concluded that HSV gB or gH can promote fusion between the virion envelope and the outer nuclear membrane. It is noteworthy that fusion associated with HSV entry requires the cooperative action of gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.
Farnsworth A, Wisner TW, Webb M, et al. Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane. Proc Nat Acad Sci US. 2007;104:10187–10192.
Correspondence: David C. Johnson at email@example.com
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Dr. Bissell is professor, Department of Pathology, Ohio State University, Columbus.