Methods for in vitro evaluating antimicrobial activity: A review

Worldwide, two billion people have been infected with hepatitis B virus (HBV), 360 million have chronic infection, and 600,000 die each year from HBV-related liver disease or hepatocellular carcinoma. This comprehensive review of hepatitis B epidemiology and vaccines focuses on definitive and influential studies and highlights current trends, policies, and directions. HBV can be transmitted vertically, through sexual or household contact, or by unsafe injections, but chronic infections acquired during infancy or childhood account for a disproportionately large share of worldwide morbidity and mortality. Vaccination against HBV infection can be started at birth and provides long-term protection against infection in more than 90% of healthy people. In the 1990s, many industrialized countries and a few less-developed countries implemented universal hepatitis B immunization and experienced measurable reductions in HBV-related disease. For example, in Taiwan, the prevalence of chronic infection in children declined by more than 90%. Many resource-poor nations have recently initiated universal hepatitis B immunization programs with assistance from the Global Alliance for Vaccines and Immunization. Further progress towards the elimination of HBV transmission will require sustainable vaccination programs with improved vaccination coverage, practical methods of measuring the impact of vaccination programs, and targeted vaccination efforts for communities at high risk of infection.

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Hepatitis B Virus Infection: Epidemiology and Vaccination

Logistical constraints associated with the collection and analysis of biological samples in community-based settings have been a significant impediment to integrative, multilevel biodemographic and biobehavioral research. However, recent methodological developments have overcome many of these constraints and have also expanded the options for incorporating biomarkers into population-based health research in international as well as domestic contexts. In particular, using dried blood spot (DBS) samples—drops of whole blood collected on filter paper from a simple finger prick— provides a minimally invasive method for collecting blood samples in nonclinical settings. After a brief discussion of biomarkers more generally, we review procedures for collecting, handling, and analyzing DBS samples. Advantages of using DBS samples—compared with venipuncture—include the relative ease and low cost of sample collection, transport, and storage. Disadvantages include requirements for assay development and validation as well as the relatively small volumes of sample. We present the results of a comprehensive literature review of published protocols for analysis of DBS samples, and we provide more detailed analysis of protocols for 45 analytes likely to be of particular relevance to population-level health research. Our objective is to provide investigators with the information they need to make informed decisions regarding the appropriateness of blood spot methods for their research interests.

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What a Drop Can Do: Dried Blood Spots as a Minimally Invasive Method for Integrating Biomarkers Into Population-Based Research

Practical HIV diagnostics are urgently needed in resource-limited settings. While HIV infection can be diagnosed using simple, rapid, lateral flow immunoassays, HIV disease staging and treatment monitoring require accurate counting of a particular white blood cell subset, the CD4+ T lymphocyte. To address the limitations of current expensive, technically demanding and/or time-consuming approaches, we have developed a simple CD4 counting microfluidic device. This device uses cell affinity chromatography operated under differential shear flow to specifically isolate CD4+ T lymphocytes with high efficiency directly from 10 microlitres of unprocessed, unlabeled whole blood. CD4 counts are obtained under an optical microscope in a rapid, simple and label-free fashion. CD4 counts determined in our device matched measurements by conventional flow cytometry among HIV-positive subjects over a wide range of absolute CD4 counts (R2 = 0.93). This CD4 counting microdevice can be used for simple, rapid and affordable CD4 counting in point-of-care and resource-limited settings.

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A microfluidic device for practical label-free CD4+ T cell counting of HIV-infected subjects

Hepatitis A virus (HAV) is spread by faecal-oral contact or ingestion of contaminated food or water. Lifelong immunity is conferred by infection or vaccination, so anti-HAV seroprevalence studies can be used to indicate which populations are susceptible to infection. Seroprevalence rates are highly correlated with socioeconomic status and access to clean water and sanitation. Increasing household income, education, water quality and quantity, sanitation, and hygiene leads to decreases in HAV prevalence. Japan, Australia, New Zealand, Canada, the United States, and most European nations have low anti-HAV rates. Although anti-HAV rates remain high in most Latin American, Asian, and Middle Eastern nations, average seroprevalence rates are declining. Surveys from Africa generally indicate no significant decline in anti-HAV rates. Because the severity of illness increases with age, populations with a high proportion of susceptible adults should consider targeted vaccination programmes.

Declining hepatitis A seroprevalence: a global review and analysis.

Background Human immunodeficiency virus (HIV) may be associated with a large number of dermatologic manifestations, which may at times constitute the presenting symptoms. These skin lesions are well delineated in the Western literature, but there is a paucity of information from the southern part of the Indian subcontinent. Objective We evaluated 833 persons with HIV to determine the types of dermatologic lesions present. Results The various lesions observed were oral candidiasis (45.0%), multidermatomal herpes zoster (11.2%), dermatophytosis of the skin (8.0%), herpes genitalis (7.7%), papular pruritic dermatitis (7.7%), staphylococcal infection of the skin (2.9%), oral hairy leukoplakia (2.3%), molluscum contagiosum (1.3%), genital warts (1.2%), and scabies (0.5%). Alopecia, intractable itching, dry skin, Addisonian pigmentation, and Kaposi's sarcoma were also noted. A correlation between the dermatologic manifestations and CD4 cell counts was found. Conclusion Although the pattern of cutaneous lesions was comparable with that from the West, there is a strikingly lower incidence of Kaposi's sarcoma.

Dermatologic manifestations among human immunodeficiency virus patients in south India.

Background & objectives: There have been very few community based studies on prevalence of hepatitis B virus (HBV) infection in India. We undertook this study to determine the prevalence of HBV infection in a southern State of India Tamil Nadu and to describe the important factors related to transmission of the virus in the community. Methods: Analysis of stored blood samples from a representative population of Tamil Nadu from an earlier community cluster survey on sexually transmitted diseases (STD) prevalence using proportionate to population size (PPS) technique was done. Serum markers of HBV viz. hepatitis B surface antigen (HbsAg) hepatitis B e antigen (Hbe Ag) and antibody to surface antigen (anti-HBs) were performed. Results: 1981 subjects were screened in the study. HbsAg prevalence was 5.7 per cent (CI 4.6-6.8) with 23.5 per cent (25/106) of these having positive HBe-antigen. Community seroprevalence (HbsAg + anti-HBs) of hepatitis B infection was 27.4 per cent (CI: 25.3-29.5) with the highest prevalence of 32.7 per cent (CI: 30.2-35.2) noted in the 15-20 yr age group. Significant independent association (OR 1.4; P=0.006) was detected with family history of exposure to HBV infection by logistic modeling. Other risk factors noted to have significant association were use of disposable needles during injection (OR 0.5; P=0.02) in men smoking (OR 3; P=0.04) and use of condom (OR 0.6; P=0.08) in women. Interpretation & conclusion: This community based study shows as high prevalence of hepatitis B infection in the state of Tamil Nadu with the highest prevalence being in the younger (15-20 yr) age group. High prevalence rate in childhood with e- antigenemia seen in 23.5 per cent of HbsAg positive subjects suggest childhood transmission. Poor injection practices and high-risk sexual behavior were found to be additional risk factors for transmission of the disease in the community. (authors)

Community prevalence of hepatitis B infection and modes of transmission in Tamil Nadu India.

Dried blood spots (DBS) on filter paper have been used as a practical method of sample collection in sero-surveillance studies of numerous diseases. DBS may be particularly useful for HIV screening in remote areas, in which unrefrigerated transfer time to a laboratory may take a number of days. In this study, we evaluate the ability to detect human immunodeficiency virus (HIV) type-1 antibodies from DBS that have been subjected to a tropical climate in southern India for 6 days. DBS were prepared from blood samples of 59 known HIV-positive individuals and 30 known HIV-negative individuals. ELISA and Western blot results from DBS that were subjected to a mean temperature of 35.8°C and humidity of 73% for 6 days had a sensitivity of 100% and 92%, respectively, and a specificity of 100% and 100%, respectively. Based on these findings, we conclude that DBS sampling could serve as a cost-effective and convenient tool for widespread HIV sero-surveillance in remote areas within tropical countries.

Dried blood spots (DBS): a valuable tool for HIV surveillance in developing/tropical countries.

We investigated 245 diarrheal stool specimens from HIV-positive subjects between January 2003 and December 2006 to determine the etiological role of coproparasites. Parasitic etiology was observed in 91 (37.1%) cases. Isospora belli (26.1%) was the most common parasite followed by Entameba histolytica/dispar (3.3%), Cryptosporidium spp. (2.9%), Giardia intestinalis (1.6%), and Strongyloides stercoralis (1.2%). Interesting trends of significant increase in the number of cases of I. belli and decline in Cryptosporidium spp. were observed during the study period.

High proportion of isosporiasis among HIV-infected patients with diarrhea in southern India.

Background and purpose Hepatitis B virus (HBV) and hepatitis C virus (HCV) dual infection accounts for a substantial proportion of liver diseases worldwide. Although the exact prevalence is not known, these viral infections are common among patients with chronic liver disease (CLD). This study was performed to determine the prevalence of HBV and HCV dual infection among patients with CLD in Chennai, India. Methods 251 patients were tested for the presence of hepatitis B surface antigen (HBsAg), immunoglobulin (Ig)-M/IgG antibody to hepatitis B core antigen (anti-HBc) and anti-HCV antibodies, and HBV-DNA and HCV-RNA by qualitative polymerase chain reaction. Results Coinfection with HCV and HBV was detected in 15 patients (5.9%), 12 of whom (80.0%) were positive for HCV-RNA and IgG anti-HBc with no evidence of HBV-DNA, while 3 HBsAg-negative patients (20.0%) were positive for HBV-DNA in addition to HCV-RNA. Liver function test profiles were significantly altered for HCV-positive patients compared with HBV-positive and HBV/HCV coinfected patients (p = 0.001). Bilirubin and alanine aminotransferase levels were significantly raised in coinfected patients compared with non-HBV, non-HCV patients (p = 0.001). Conclusions The results demonstrated that HBV was predominantly associated with underlying CLD among this group of patients in India and suggest that HBV coinfection in HCV-infected patients should not be excluded by negative HBsAg status alone.

S P Thyagarajan

Papers

Dermatologic manifestations among human immunodeficiency virus patients in south India.

Community prevalence of hepatitis B infection and modes of transmission in Tamil Nadu India.

Dried blood spots (DBS): a valuable tool for HIV surveillance in developing/tropical countries.

High proportion of isosporiasis among HIV-infected patients with diarrhea in southern India.

Hepatitis B virus and hepatitis C virus dual infection among patients with chronic liver disease.