Genetic stability of a dengue vaccine based on chimeric yellow fever/dengue viruses.

A tetravalent dengue vaccine based on four live, attenuated, chimeric viruses (CYD1-4), constructed by replacing the genes coding for premembrane (prM) and envelope (E) proteins of the yellow fever (YF)-17D vaccine strain with those of the four serotypes of dengue virus, is in clinical phase III evaluation. We assessed the vaccine's genetic stability by fully sequencing each vaccine virus throughout the development and manufacturing process. The four viruses displayed complete genetic stability, with no change from premaster seed lots to bulk lots. When pursuing the virus growth beyond bulk lots, a few genetic variations were observed. Usually both the initial nucleotide and the new one persisted, and mutations appeared after a relatively high number of virus duplication cycles (65-200, depending on position). Variations were concentrated in the prM-E and non-structural (NS)4B regions. PrM-E variations had no impact on lysis-plaque size or neurovirulence in mice. None of the variations located in the YF-17D-derived genes corresponded with reversion to the wild-type Yellow Fever sequence. Variations in NS4B likely reflect virus adaptation to Vero cells growth. A low to undetectable viremia has been reported previously [1-3] in vaccinated non-human and human primates. Combined with the data reported here about the genetic stability of the vaccine strains, the probability of in vivo emergence of mutant viruses appears very low.

Standardized quantitative RT-PCR assays for quantitation of yellow fever and chimeric yellow fever-dengue vaccines.

Yellow fever-dengue chimeras (CYDs) are being developed currently as live tetravalent dengue vaccine candidates. Specific quantitative assays are needed to evaluate the viral load of each serotype in vaccine batches and biological samples. A quantitative real-time RT-PCR (qRT-PCR) system was developed comprising five one-step qRT-PCRs targeting the E/NS1 junction of each chimera, or the NS5 gene in the yellow fever backbone. Each assay was standardized using in vitro transcribed RNA qualified according to its size and purity, and precisely quantified. A non RNA-extracted virus sample was introduced as external quality control (EQC), as well as 2 extraction controls consisting of 2 doses, 40 and 4,000 GEQ (genomic equivalents), of this EQC extracted in parallel to the samples. Between 6 and 10 GEQ/reaction were reproducibly measured with all assays and similar titers were obtained with the two methods when chimeric virus samples were quantified with the E/NS1- or the NS5-specific assays. Reproducibility of RNA extraction was ensured by automation of the process (yield>or=50%), and infectious virus was isolated in >or=80% of PCR-positive sera from immune monkeys.

The efficiency of the sets and the cuscore techniques under biased baseline rates.

Statistical techniques used for surveillance of disease incidence rates are generally based on the assumption of known baseline rate of the disease monitored, whereas actually it is an estimate obtained from a large sample. As a result, the time interval until true or false alarm is shorter or longer than assumed. In this study, we evaluate the performance of the sets and of the cuscore techniques when the estimate of the baseline rate is biased. We evaluate the effect of an underestimated baseline rate with respect to frequency of false alarms and to that of an over estimated rate with respect to the delay until elicitation of a true alarm. We evaluate the effects of 5 per cent and 10 per cent bias in the estimated baseline rate for specified conditions associated with sparse data. The results show that the effect of plus or minus 5 per cent bias in the estimate are moderate and those of 10 per cent are substantial. In general, the effect of an overestimated baseline rate is greater on the sets technique than it is on the cuscore technique and the effect of an underestimated rate is greater on the cuscore technique than it is on the sets technique. However, the differences between the two techniques are small on both perspectives. The two methods differ also with respect to the expected time until true alarm when the specified baseline rate is unbiased. The sets technique is the more efficient in detecting a two-fold increased rate when the number of diagnoses expected annually (E(X)) is less than 1.62, and the cuscore is the more efficient technique when E(X) > 1.62. We use the term 'turning point' to define the regions in which the sets technique and the cuscore techniques are preferred. With an estimated baseline rate that is 5 per cent higher than the actual rate, the turning point falls from 1.62 to 1.45 when the rate is twice the baseline rate, and from 5.75 to 4.34 when the rate is triple the baseline rate.

Motor neuron disease on Guam: geographic and familial occurrence, 1956-85.

We investigated the geographic and familial occurrence of motor neuron disease (MND) on Guam, and then considered etiologic hypotheses related to cycad use and metal intoxication. The research was based on 303 Chamorros from Guam and 3 Chamorros from other Mariana Islands, all with MND onset on Guam during 1956-85. Inarajan and Umatac, two southern districts, each had, for both sexes combined, an average incidence rate significantly higher than the corresponding overall rate for Guam. Also, for each sex, geographic patterns of incidence were significantly related to 1) socioeconomic level (men only), 2) cycasin concentrations in cycad flour samples (men and women), 3) iron concentrations in water samples (men and women), 4) silicon concentrations in water samples (men only), and 5) cobalt and nickel concentrations in soil samples (men and women). The MND risk in susceptible sibships was about 7-28 times greater than that in the general population. The cycad hypothesis conforms somewhat better than the metal intoxication hypothesis with the data presented.

Motor neuron disease on Guam: temporal occurrence, 1941-85.

Using a case registry, we investigated the temporal occurrence of motor neuron disease (MND) on Guam. MND with onset during 1941-85 was documented in 434 Chamorros and 9 non-Chamorro migrants who had lived on Guam before onset. Increased median age at onset and decreased age-adjusted incidence rates (since the early 1960s) were observed for Chamorros of both sexes. Our evidence about MND on Guam is consistent with: 1) The latent period duration has varied from years to decades; 2) With time, the exposure period or latent period, or both, have lengthened; 3) The high risk of acquiring the condition has been reduced since, at least, the early 1950s, and the most recent years of meaningful risk were the early to middle 1960s; 4) The critical age for acquiring the condition is in adolescence and adulthood; 5) Change of environment from Guam to overseas during childhood resulted in decreased risk of acquiring the condition.

Analysing post-alarm data in a monitoring system in order to accept or reject the alarm.

In a disease monitoring system embracing several diseases and communities, alarms indicating a significant increase in disease incidence will frequently occur even though the false alarm rate has been set quite low. An approach is presented by which an alarm is determined as either confirmed or rejected according to data observed subsequent to the alarm. The suggested procedure is expected to weed out a substantial proportion (say about 75 per cent) of the false alarms at the expense of some delay in detecting a true alarm.

Mainstream and environmental tobacco smoke.

Environmental tobacco smoke (ETS) is derived from cigarette smoldering and active smoker exhalation. Its composition displays broad quantitative differences and redistributions between gas and respirable suspended particulate (RSP) phases when compared with the mainstream smoke (MSS) that smokers puff. This is because of different generation conditions and because ETS is diluted and ages vastly more than MSS. Such differences prevent a direct comparison of MSS and ETS and their biologic activities. However, even assuming similarities on an equal mass basis, ETS-RSP inhaled doses are estimated to be between 10,000- and 100,000-fold less than estimated average MSS-RSP doses for active smokers. Differences in effective gas phase doses are expected to be of similar magnitude. Thus the average person exposed to ETS would retain an annual dose analogous to the active MSS smoking of considerably less than one cigarette dispersed over a 1-year period. By contrast, consistent epidemiologic data indicate that active smoking of some 4-5 cigarettes per day may not be associated with a significantly increased risk of lung cancer. Similar indications also obtain for cardiovascular and respiratory diseases. Since average doses of ETS to nonsmoking subjects in epidemiologic studies are several thousand times less than this reported intake level, the marginal relative risks of lung cancer and other diseases attributed to ETS in some epidemiologic studies are likely to be statistical artifacts, derived from unaccounted confounders and unavoidable bias.

Geographic patterns of parkinsonism-dementia complex on Guam. 1956 through 1985.

Average annual age-adjusted incidence rates of parkinsonism-dementia complex were obtained for the 19 election districts of Guam from 1956 through 1985. The highest rates were found in the southern and central districts, and the lowest rates were found in the northern and western districts. Geographic and temporal patterns of incidence were associated with socioeconomic status but not with geochemical factors. The risk of parkinsonism-dementia complex in susceptible sibships was much higher than that in the general population--even in districts with the highest incidence rates, but especially in districts with the lowest incidence rates. Our evidence tends to support the hypothesis that multiple factors linked to cycad use play an important role in the cause of PDC. Hypotheses related to metal exposure and simple genetic factors were unsatisfactory explanations for the epidemiologic patterns observed.

Patterns of acquiring parkinsonism-dementia complex on Guam. 1944 through 1985.

For 1944 through 1985, documented clinical descriptions compatible with a diagnosis of parkinsonism-dementia complex were found in 363 Chamorros (including mixed Chamorros) and three Filipino immigrants who had lived on Guam before onset. Downward trends in age-adjusted incidence rates and upward trends in age at onset were seen for Chamorros of both sexes. Since 1980, new cases have occurred only among persons over 50 years of age, whereas a younger age at onset had been noted in the past. Evidence suggests that the length of the latent period varied widely (up to decades) and increased with time, the risk of acquiring parkinsonism-dementia complex had declined since the late 1950s, and the critical age of exposure to an unknown factor in the environment on Guam appears to have been during adolescence or adulthood.

An analysis of two recent epidemiologic reports in the New England Journal of Medicine associating breast cancer in women with moderate alcohol consumption.

Two cohort studies showed an association between moderate alcohol consumption and breast cancer, with an apparent relative risk of 1.50. Drinkers and nondrinkers already differed in important ways at the initiation of these two studies, precluding a causal interpretation of the associations found. Women in need of the relaxing effect of alcohol may indeed be the group at higher risk for breast cancer. Inasmuch as the studies did not inquire into other cancers, morbidities, mortalities, or total mortality, they leave open the possibility that, on balance, moderate alcohol consumption is beneficial.