Vaccines against enteric infections for the developing world

Since the first licensure of the Sabin oral polio vaccine more than 50 years ago, only eight enteric vaccines have been licensed for four disease indications, and all are given orally. While mucosal vaccines offer programmatically attractive tools for facilitating vaccine deployment, their development remains hampered by several factors:

• — limited knowledge regarding the properties of the gut immune system during early life;
• — lack of mucosal adjuvants, limiting mucosal vaccine development to live-attenuated or killed whole virus and bacterial vaccines;
• — lack of correlates/surrogates of mucosal immune protection; and
• — limited knowledge of the factors contributing to oral vaccine underperformance in children from developing countries.

There are now reasons to believe that the development of safe and effective mucosal adjuvants and of programmatically sound intervention strategies could enhance the efficacy of current and next-generation enteric vaccines, especially in lesser developed countries which are often co-endemic for enteric infections and malnutrition. These vaccines must be safe and affordable for the world''s poorest, confer long-term protection and herd immunity, and must be able to contain epidemics.     

New methods for the diagnosis of enteric infections

Gastrointestinal disease remains a major cause of mortality and morbidity throughout the world. Recently, a number of viral, bacterial, and protozoan agents have been identified which can cause a range of gastrointestinal disorders. The effective management of these diseases requires the prompt identification of the infecting micro-organism and the early institution of preventative and therapeutic interventions. The detection of infecting microorganisms in fecal and intestinal fluids presents a particular challenge to the diagnostic microbiologist. Cultivation can be difficult due to the fastidious nature of the microorganisms and the presence of cytotoxic materials in the specimen. In the past, immunoassays have been used for the detection of some microorganisms. However, immunoassays have limited sensitivity and cannot detect all infecting microorganisms. Recently, nucleic acid amplification techniques have been developed for the direct detection of pathogenic microbial DNA and RNA in human body fluids. We have found that these methods can be applied for the accurate detection of intestinal vlruses provided that inhibitors of enzymatic amplification are removed from the sample. Using affinity binding purification and non-isotopic DNA measurement techniques, we have developed sensitive and specific assays for the quantitation of a wide range of infecting microorganisms in intestinal fluids. Nucleic acid amplification provides a Abstract continued on next page unique tool for the study of enteric pathogens and for the development of strategies for their eventual elimination from the human environment.This paper was presented at the IUMS Symposium on New Developments in Diagnosis and Control of Infectious Diseases held in conjunction with the Eighth International Congress of Virology, Berlin, Germany, 24–31 August 1990.     

A rapid plant assay for the parasponia-rhizobium symbiosis

A plant assay was devised which enabled the rapid and reliable assessment of nodulation and nitrogen fixation in the symbiosis formed between Parasponia andersonii and Parasponia Rhizobium strain CP279. Seed germination was obtained in 4 days with 87% germination after 13 days. An agar plate plant assay allowed nodulation of all replicates in 7–8 days with nitrogen fixation established at 21 days as measured by acetylene reduction. Nodules could also be assessed for effectiveness by a visual comparison between inoculated and uniculated plants or by differences in total plant fresh wt. after 6 weeks of growth.     

Development of a fluorescent and colorimetric detection methods-based protein microarray for serodiagnosis of TORCH infections

We developed a protein microarray methodology that has the ability of serodiagnosis of IgM antibodies directed against TORCH pathogens. Six chemical surface modifications were validated by a dimension atomic force microscope (AFM) and contact angle measurement, agarose modified surface of which offered an appropriate platform for detecting IgM antibody. Further, signal amplification sensitivities on agarose modified microarrays were detected by Cy3-labeled biotin-streptavidin and immunogold-based assays. The detection limits of IgM antibody on the microarrays were 0.48 and 0.24mug/ml, quantitatively equal to 0.25 and 12.5pg, respectively, on each spot as ascertained by the two assays. Satisfactory linear correlations between the signal intensity and the logarithm of the IgM concentration were obtained. Finally, 60 serum samples characterized by a commercial ELISA were evaluated by the protein microarray. There were good concordances between the results of the protein microarray and ELISA assay for sorting of the TORCH infected sera (95.0% by fluorescence-based assay and 96.7% by immunogold-based assay). Clearly, the potential application of this protein microarray format facilitates clinical detection of not only the antibodies directed against TORCH pathogens but also other autoimmune diseases.     

Use of loading doses of bifidumbacterin forte for treatment of patients with acute enteric infections

The clinical observation of patients with acute enteric infections (AEI), treated with loading doses of Bifidumbacterin forte during the first 2 days of the disease, was carried out. The preparation was shown to produce a positive effect on the course of AEI: salmonellosis, alimentary toxicoinfections, acute, dysentery. The early decrease of the manifestations of intoxication, pain syndrome, diarrhea, as well as the acceleration of convalescence in comparison with standard treatment, were noted. The most essential dynamics was registered in salmonellosis patients. The analysis of clinical results allowed to recommend the use of loading doses of Bifidumbacterin forte, a probiotic with high colonization potential to normalize the microbiocenosis of the intestine in AEI.     

A prevalence survey for zoonotic enteric bacteria in a research monkey colony with specific emphasis on the occurrence of enteric Yersinia

Transmissible pathogenic and opportunistic zoonotic enteric bacteria comprise a recognized occupational health threat to exposed humans from non-human primates (NHPs). In an effort to evaluate the occurrence of selected enteric organisms with zoonotic and biohazard potential in a research colony setting, we performed a prevalence study examining 61 juvenile and young adult rhesus macaques participating in a transplant immunology project. Primary emphasis was directed specifically to detection of pathogenic enteric Yersinia, less well-documented and reported NHP pathogens possessing recognized significant human disease potential. NHPs were surveyed by rectal culture during routine health monitoring on three separate occasions, and samples incubated using appropriate media and specific selective culture methods. Enteric organisms potentially transmissible to humans were subcultured and identified to genus and species. Significant human pathogens of the Salmonella/Shigella, Campylobacter, and enteric Yersinia groups were not isolated throughout the survey, suggesting prevalence of these organisms may generally be quite low.     

胶体金渗滤法检测贝氏柯克斯体的研究

本文建立一种快速、敏感、适于基层应用的贝氏柯克斯体（俗称Ｑ热立克次体）的检测方法。将Ｑ热立克次体多克隆抗体点于硝酸膜上,用以捕获待检标本中的Ｑ热立克次体抗原,通过胶体金标记的鼠抗Ｑ热立克次体单克隆抗体直接显色,阳性者出现红色斑点。结果表明,用该法检测Ｑ热立克次休实验感染豚鼠血液,小鼠肝或脾,蜱血淋巴等标本取得了较满意的结果,整个过程仅需５－６分钟。与其他病原体无交叉反应,敏感度不低于５０ｎｇ立克次     

Evaluation of vaccines against enteric infections: a clinical and public health research agenda for developing countries

Enteric infections are a major cause of morbidity and mortality in developing countries. To date, vaccines have played a limited role in public health efforts to control enteric infections. Licensed vaccines exist for cholera and typhoid, but these vaccines are used primarily for travellers; and there are two internationally licensed vaccines for rotavirus, but they are mainly used in affluent countries. The reasons that enteric vaccines are little used in developing countries are multiple, and certainly include financial and political constraints. Also important is the need for more cogent evidence on the performance of enteric vaccines in developing country populations. A partial inventory of research questions would include: (i) does the vaccine perform well in the most relevant settings? (ii) does the vaccine perform well in all epidemiologically relevant age groups? (iii) is there adequate evidence of vaccine safety once the vaccines have been deployed in developing countries? (iv) how effective is the vaccine when given in conjunction with non-vaccine cointerventions? (v) what is the level of vaccine protection against all relevant outcomes? and (vi) what is the expected population level of vaccine protection, including both direct and herd vaccine protective effects? Provision of evidence addressing these questions will help expand the use of enteric vaccines in developing countries.     

Assessment of antibody responses to antigens of Mycobacterium tuberculosis and Cysticercus cellulosae in cerebrospinal fluid of chronic meningitis patients for definitive diagnosis as TBM/NCC by passive hemagglutination and immunoblot assays

Tanned sheep erythrocytes stabilized with pyruvic aldehyde and glutaraldehyde, called double-aldehyde-stabilized cells, were used to standardize passive hemagglutination assay (PHA) for detection of antibody responses to sonicate extract of Mycobacterium tuberculosis and Cysticercus cellulosae soluble antigens. PHA was performed in the following groups of cerebrospinal fluid (CSF) samples: group I - chronic infections of the central nervous system with the possible diagnosis of tuberculous meningitis (TBM), tuberculoma and neurocysticercosis (NCC) (n=88), and group II - controls which included (a) non-infectious non-neurological conditions (n=30), (b) infectious neurological conditions (n=21) and (c) non-infectious neurological conditions (n=133). PHA could detect anti-mycobacterial antibodies at the sensitivity level of 80.76% with a specificity of 92.4% and anti-cysticercal antibodies with a sensitivity of 100% and specificity of 92.94%. However, in 6.33% (i.e. 14/221) of group I and group II (c) CSFs both anti-mycobacterial and anti-cysticercal antibodies were detected. Immunoblot analysis of CSFs derived from TBM patients reacted predominantly to 120-kDa, 96-kDa, 65-kDa, 38-kDa, 26-kDa, 23-kDa, 19-kDa and 12-14-kDa and 4-6-kDa antigens of M. tuberculosis sonicate extract (MTSE), whilst CSFs of proven NCC reacted to >110-kDa, 96-kDa, 80-kDa, 66-68-kDa, 52-kDa and 26-28-kDa antigens of porcine whole cyst sonicate extract (PCSE). On immunoblot analysis, some of the CSFs of TBM patients were PHA positive for both MTSE and PCSE showed antibody reactivity to 70-kDa and 10-kDa antigens of C. cellulosae. Similarly CSF antibody of some Guillain Barre syndrome and myeloradiculopathy patients reacted with cysticercal antigens. But per se no cross-reactivity between MTSE and anti-cysticercal antibodies and vice-versa were observed. However, findings of this study should alert laboratory personnel especially in endemic areas to be extra careful in interpretation of antibody detection results.     

2009～2010年上海地区急性呼吸道感染病毒病原谱分析

调查2009~2010年上海地区人群急性呼吸道感染(ARTI)的病毒性病原,探讨2009甲型H1N1流感暴发背景下呼吸道感染病毒病原谱的构成。采用套式多重反转录-聚合酶链反应(RT-PCR)和实时荧光定量RT-PCR方法,对来自2 044例患者的2 044份标本(包括2 005份鼻咽拭子和39份肺泡灌洗液),同时检测腺病毒(ADV)、副流感病毒(PIV)、甲型流感病毒(FluA)、乙型流感病毒(FluB)、微小核糖核酸病毒、呼吸道合胞病毒(RSV)、人偏肺病毒(hMPV)、冠状病毒(CoV)和人博卡病毒(HBoV)。其中,656(32.09%)份标本经呼吸道病毒检测为阳性,52份标本为双重感染。FluA检出率最高(13.36%),其后依次为微小核糖核酸病毒(10.23%)、FluB(4.84%)、ADV(1.96%)、PIV(1.76%)、RSV(1.32%)、CoV(0.59%)、hMPV(0.39%)和HBoV(0.20%)。但各月病毒检出率分布不均,2009和2010年呼吸道病毒检出率高峰出现在当年11月(53.07%和65.59%),低谷都出现在当年5月,且2009年5~9月的病毒检出率高于2010年同期(32.02%vs15.38%,P<0.05)。其中,2009甲型H1N1流感暴发导致2009年10月~2010年1月2009甲型H1N1流感病毒占当月检出FluA的100%,2009年6~9月也占当月检出FluA的较高比率,依次为90.91%(20/22)、75.00%(15/20)、48.00%(12/25)和56.25%(18/32)。比较甲型H3N2流感病毒和2009甲型H1N1流感病毒分别在上呼吸道感染(URTI)和下呼吸道感染(LRTI)中的检出率,无统计学差异(URTI,85.29%vs76.61%;LRTI,14.71%vs23.39%;P>0.05)。呼吸道病毒检出率还与年龄相关,0~4岁组和5~14岁组病毒检出率高于其他年龄组。在0~4岁及≥65岁组中,微小核糖核酸病毒检出率最高,FluA次之;其余年龄组中FluA检出率最高。混合感染中15岁以下儿童占50%(26/52),微小核糖核酸病毒与其他病毒混合感染占84.62%(44/52)。本研究表明,上海地区2009~2010年FluA是最常见的急性呼吸道感染病原,2009甲型H1N1流感病毒成为2009年FluA的优势亚型。微小核糖核酸病毒是混合感染中最常见的病原。结果提示,应长期监测主要呼吸道病毒的活动水平,并加强对微小核糖核酸病毒流行病学和致病性的研究。     

A rapid assay for assessment of sphingosine kinase inhibitors and substrates

Sphingosine kinases (SphKs) catalyze the transfer of phosphate from adenosine triphosphate (ATP) to sphingosine to generate sphingosine 1-phosphate (S1P), an important bioactive lipid molecule that mediates a diverse range of cell signaling processes. The conventional assay of SphK enzymatic activity uses [γ-³²P]ATP and sphingosine as substrates, with the radiolabeled S1P product recovered by organic extraction, displayed by thin layer chromatography, and quantified by liquid scintillation counting. Although this assay is sensitive and accurate, it is slow and labor-intensive; thus, it precludes the simultaneous screening of more than a few inhibitor compounds. Here we describe a 96-well assay for SphKs that is rapid and reproducible. Our method, which takes advantage of the limited solubility of S1P, detects radioactive S1P adhering to the plate by scintillation proximity counting. Our procedure obviates extraction into organic solvents, postreaction transfers, and chromatography. Furthermore, our assay enables assessment of both inhibitors and substrates, and it can detect endogenous SphK activity in cell and tissue extracts. The SphK kinetic parameter, K_m, and the K_i values of inhibitors determined with our assay and the conventional assay were indistinguishable. These results document that our assay is well-suited for the screening of chemical libraries of SphK inhibitors.     

Imperfect vaccines and the evolution of pathogens causing acute infections in vertebrates

A study by Gandon et al. (2001) considered the potential ways pathogens may evolve in response to vaccination with imperfect vaccines. In this paper, by focusing on acute infections of vertebrate hosts, we examine whether imperfect vaccines that do not completely block a pathogen's replication (antigrowth) or transmission (antitransmission) may lead to evolution of more or less virulent pathogen strains. To address this question, we use models of the within-host dynamics of the pathogen and the host's immune responses. One advantage of the use of this within-host approach is that vaccination can be easily incorporated in the models and the trade-offs between pathogen transmissibility, host recovery, and virulence that drive evolution of pathogens in these models can be easily estimated. We find that the use of either antigrowth or antitransmission vaccines leads to the evolution of pathogens with an increased within-host growth rate; infection of unvaccinated hosts with such evolved pathogens results in high host mortality and low pathogen transmission. Vaccination of only a fraction of hosts with antigrowth vaccines may prevent pathogens from evolving high virulence due to pathogen adaptation to unvaccinated hosts and thus protection of vaccinated hosts from pathogen-induced disease. In contrast, antitransmission vaccines may be beneficial only if they are effective enough to cause pathogen extinction. Our results suggest that particular mechanisms of action of vaccines and their efficacy are crucial in predicting longterm evolutionary consequences of the use of imperfect vaccines.     

Evaluation of rapid immunochromatographic assay kit for HBsAg-screening using whole blood

A rapid immunochromatographic assay kit using whole blood to screen hepatitis B surface antigen was developed and evaluated by using sera from 240 patients. The reference diagnosis was based on the results obtained with GENEDIA Anti-HBs Rapid kit which is very similar to the above kit except for the use of serum. The test demonstrated a good correlation with the reference immunochromatographic assay kit, that is, the sensitivity and the specificity of the kit was 100%, respectively. The rapid test kit using whole blood should be more convenient and useful for the diagnosis of hepatitis B virus because the kit does not need machines and time to prepare serum. In addition, this kit is safe from inadvertent infection during sample treatment because the blood is sterilized with hydrogen peroxide, eliminates the procedure required to prepare serum and reduces the possibility of exposure to infectious agents.     

An improved syncytia infectivity assay for the bovine leukemia virus

Summary Several factors that influence the sensitivity of the syncytia infectivity assay for the bovine leukemia virus (BLV) and BLV-infected lymphocytes have been examined. The use of early-passage indicator bovine embryonic spleen (BESP) cells and their pretreatment with diethylamino-ethyl-dextran (DEAE-D) was essential for optimal sensitivity. Polybrene was less effective than DEAE-D. The combination of DEAE-D and polybrene was more effective than DEAE-D alone when BLV-infected leukocytes were used as the inoculum, but not when the inoculum was a cell-free BLV preparation. Using BESP cell passages 4 to 11 as indicators, reproducible titers were obtained when aliquots of the same virus stock were assayed at different times after freezer storage. When assaying peripheral blood lymphocytes from infected cattle, optimal syncytia responses were observed consistently by inoculating 5×10⁶ viable lymphocytes per 60-mm Falcon dish. Centrifugation of peripheral blood leukocytes from BLV-infected cattle in discontinuous bovine serum albumin gradients can be used to separate a subpopulation of infected lymphocytes. Use of this subpopulation as the inoculum, rather than unseparated buffy-coat leukocytes, greatly increases the sensitivity of the syncytia infectivity assay. This work was supported in part by USPHS Grant 1-PO 1-CA-14193-03, Pennsylvania Department of Agriculture Grant ME4, and USDA Cooperative Agreement 12-14-100-10, 675 (45).     

A rapid real-time recombinase polymerase amplification assay for diagnosis of acute hepatopancreatic necrosis disease in shrimp

Acute hepatopancreatic necrosis disease(AHPND)is an emerging disease in the shrimp farming industry with a high mortality rate that causes serious economic loss...     

A rapid and sensitive assay for determination of cholesterol in membrane lipid extracts

A commercially available enzymatic assay (Boehringer Monotest) was modified to allow a rapid and sensitive determination of cholesterol in membrane lipid extracts. This was achieved by adding 0.5% Triton X-100 to the reagent solution. The detergent did not interfere with the assay. The relationship between the amount of cholesterol per assay and the absorbance at 500 nm was linear up to 100 μg. The recovery in the assay was better than 95%. The assay was applied to the determination of cholesterol in erythrocyte membrane lipid extracts.     

Novel recombinant RD2- and RD11-encoded Mycobacterium tuberculosis antigens are potential candidates for diagnosis of tuberculosis infections in BCG-vaccinated individuals

Proteins encoded by region of deletions (RD) of Mycobacterium tuberculosis are useful in development of vaccines and diagnostic reagents. In the present study, six M. tuberculosis genes from RD2 and RD11, rv1978, nrdf1, mpt64, cfp-21, ppe57 and ppe59, were cloned and overexpressed in Escherichia coli. All six purified recombinant proteins could distinguish tuberculosis (TB) patients and latent TB infected subjects (LTBI), or called subclinical TB infection, from BCG-vaccinated healthy controls by T-cell IFN-γ releasing ELISPOT. ELISPOT of Rv1978, NrdF1, Mpt64, CFP-21, Ppe57 and Ppe59 achieved sensitivities of 59%, 60%, 82%, 48%, 59% and 47% respectively in the detection of active TB and specificities of 94%, 90%, 76%, 93%, 100% and 93% respectively in BCG-vaccinated healthy controls. Combination of Ppe57 or NrdF1 with early secreted antigen target 6 (ESAT-6) or 10-kDa culture filtrate protein (CFP-10) in the IFN-γ releasing ESLIPOT assay could increase the sensitivities in detecting active TB, for ESAT-6 from 82.1% to 85.7% or 92.9% (P = 0.5 or 0.03, respectively) and for CFP-10 from 67.9% to 78.6% or 83.9%, respectively (both P < 0.05). The high sensitivities, specificities and promising antigenic combination of NrdF1 and Ppe57 in detection of TB in BCG-vaccinated controls suggest their potential application in TB diagnosis.