COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

The use of a "direct PCR" DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme\u27s resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens

Species-Specific Serological Detection for Schistosomiasis by Serine Protease Inhibitor (SERPIN) in Multiplex Assay

Background: Both Schistosoma mansoni and Schistosoma haematobium cause schistosomiasis in sub-Saharan Africa. We assessed the diagnostic value of selected Schistosoma antigens for the development of a multiplex serological immunoassay for sero-epidemiological surveillance. Methodology/Principal Findings: Diagnostic ability of recombinant antigens from S. mansoni and S. haematobium was assessed by Luminex multiplex immunoassay using plasma from school children in two areas of Kenya, endemic for different species of schistosomiasis. S. mansoni serine protease inhibitor (SERPIN) and Sm-RP26 showed significantly higher reactivity to patient plasma as compared to the control group. Sm-Filamin, Sm-GAPDH, Sm-GST, Sm-LAP1, Sm-LAP2, Sm-Sm31, Sm-Sm32 and Sm-Tropomyosin did not show difference in reactivity between S. mansoni infected and uninfected pupils. Sm-RP26 was cross-reactive to plasma from S. haematobium patients, whereas Sm-SERPIN was species-specific. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. ROC analysis for Sm-RP26, Sm-SERPIN and Sh-SERPIN showed AUC values of 0.833, 0.888 and 0.947, respectively. Using Spearman’s rank correlation coefficient analysis, we also found significant positive correlation between the number of excreted eggs and median fluorescence intensity (MFI) from the multiplex immunoassays for Sm-SERPIN (ρ = 0.430, p-value = 0.003) and Sh-SERPIN (ρ = 0.433, p-value = 0.006). Conclusions/Significance: Sm-SERPIN is a promising species-specific diagnostic antigen. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. SERPINs showed correlation with the number of excreted eggs. These indicate prospects for inclusion of SERPINs in the multiplex serological immunoassay system

Influence of socioeconomic factors on medically unnecessary ambulance calls

<p>Abstract</p> <p>Background</p> <p>Unnecessary ambulance use has become a socioeconomic problem in Japan. We investigated the possible relations between socioeconomic factors and medically unnecessary ambulance calls, and we estimated the incremental demand for unnecessary ambulance use produced by socioeconomic factors.</p> <p>Methods</p> <p>We conducted a self-administered questionnaire-based survey targeting residents of Yokohama, Japan. The questionnaire included questions pertaining to socioeconomic characteristics, dichotomous choice method questions pertaining to ambulance calls in hypothetical nonemergency situations, and questions on the city's emergency medical system. The probit model was used to analyze the data.</p> <p>Results</p> <p>A total of 2,029 out of 3,363 targeted recipients completed the questionnaire (response rate, 60.3%). Probit regression analyses showed that several demographic and socioeconomic factors influence the decision to call an ambulance. Male respondents were more apt than female respondents to state that they would call an ambulance in nonemergency situations (p < 0.05). Age was an important factor influencing the hypothetical decision to call an ambulance (p < 0.05); elderly persons were more apt than younger persons to state that they would call an ambulance. Possession of a car and hesitation to use an ambulance negatively influenced the hypothetical decision to call an ambulance (p < 0.05). Persons who do not have a car were more likely than those with a car to state that they would call an ambulance in unnecessary situations.</p> <p>Conclusion</p> <p>Results of the study suggest that several socioeconomic factors, i.e., age, gender, household income, and possession of a car, influence a person's decision to call an ambulance in nonemergency situations. Hesitation to use an ambulance and knowledge of the city's primary emergency medical center are likely to be important factors limiting ambulance overuse. It was estimated that unnecessary ambulance use is increased approximately 10% to 20% by socioeconomic factors.</p

Serological Surveillance Development for Tropical Infectious Diseases Using Simultaneous Microsphere-Based Multiplex Assays and Finite Mixture Models

Background:A strategy to combat infectious diseases, including neglected tropical diseases (NTDs), will depend on the development of reliable epidemiological surveillance methods. To establish a simple and practical seroprevalence detection system, we developed a microsphere-based multiplex immunoassay system and evaluated utility using samples obtained in Kenya.Methods:We developed a microsphere-based immuno-assay system to simultaneously measure the individual levels of plasma antibody (IgG) against 8 antigens derived from 6 pathogens: Entamoeba histolytica (C-IgL), Leishmania donovani (KRP42), Toxoplasma gondii (SAG1), Wuchereria bancrofti (SXP1), HIV (gag, gp120 and gp41), and Vibrio cholerae (cholera toxin). The assay system was validated using appropriate control samples. The assay system was applied for 3411 blood samples collected from the general population randomly selected from two health and demographic surveillance system (HDSS) cohorts in the coastal and western regions of Kenya. The immunoassay values distribution for each antigen was mathematically defined by a finite mixture model, and cut-off values were optimized.Findings:Sensitivities and specificities for each antigen ranged between 71 and 100%. Seroprevalences for each pathogen from the Kwale and Mbita HDSS sites (respectively) were as follows: HIV, 3.0% and 20.1%; L. donovani, 12.6% and 17.3%; E. histolytica, 12.8% and 16.6%; and T. gondii, 30.9% and 28.2%. Seroprevalences of W. bancrofti and V. cholerae showed relatively high figures, especially among children. The results might be affected by immunological cross reactions between W. bancrofti-SXP1 and other parasitic infections; and cholera toxin and the enterotoxigenic E. coli (ETEC), respectively.Interpretation:A microsphere-based multi-serological assay system can provide an opportunity to comprehensively grasp epidemiological features for NTDs. By adding pathogens and antigens of interest, optimized made-to-order high-quality programs can be established to utilize limited resources to effectively control NTDs in Africa

Comparacao de seis polimerases de DNA disponiveis comercialmente para o PCR direto

SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.RESUMO O propósito deste estudo foi avaliar 6 polimerases de DNA disponíveis comercialmente que são resistentes aos inibidores do PCR para uma amplificação potencial de DNA de amostras de sangue total. O DNA genômico do parasita humano da malária, Plasmodium falciparum, foi analisado sob condições que incluíram os componentes inibidores do sangue extraído de sangue ressacado em papel de filtro. Nossos resultados sugerem que a polimerase KOD FX DNA é superior a outras polimerases

Spatial Distributions of HIV Infection in an Endemic Area of Western Kenya: Guiding Information for Localized HIV Control and Prevention.

HIV is still a major health problem in developing countries. Even though high HIV-risk-taking behaviors have been reported in African fishing villages, local distribution patterns of HIV infection in the communities surrounding these villages have not been thoroughly analyzed. The objective of this study was to investigate the geographical distribution patterns of HIV infection in communities surrounding African fishing villages. In 2011, we applied age- and sex-stratified random sampling to collect 1,957 blood samples from 42,617 individuals registered in the Health and Demographic Surveillance System in Mbita, which is located on the shore of Lake Victoria in western Kenya. We used these samples to evaluate existing antibody detection assays for several infectious diseases, including HIV antibody titers. Based on the results of the assays, we evaluated the prevalence of HIV infection according to sex, age, and altitude of participating households. We also used Kulldorff's spatial scan statistic to test for HIV clustering in the study area. The prevalence of HIV at our study site was 25.3%. Compared with the younger age group (15-19 years), adults aged 30-34 years were 6.71 times more likely to be HIV-positive, and the estimated HIV-positive population among women was 1.43 times larger than among men. Kulldorff's spatial scan statistic detected one marginally significant (P = 0.055) HIV-positive and one significant HIV-negative cluster (P = 0.047) in the study area. These results suggest a homogeneous HIV distribution in the communities surrounding fishing villages. In addition to individual behavior, more complex and diverse factors related to the social and cultural environment can contribute to a homogeneous distribution pattern of HIV infection outside of African fishing villages. To reduce rates of transmission in HIV-endemic areas, HIV prevention and control programs optimized for the local environment need to be developed