Transcriptional Analysis of Rickettsia prowazekii Invasion Gene Homolog (invA) during Host Cell Infection

An invasion gene homolog, invA, of Rickettsia prowazekii has recently been identified to encode a member of the Nudix hydrolase subfamily which acts specifically on dinucleoside oligophosphates (Np(n)N; n ≥ 5), a group of cellular signaling molecules known as alarmones. InvA is thought to enhance intracellular survival by regulating stress-induced toxic nucleotide levels during rickettsial infection. To further characterize the physiological function of InvA, the gene expression pattern during various stages of rickettsial intracellular growth was investigated. Using semiquantitative reverse transcription-PCR (RT-PCR) and real-time fluorescent probe-based quantitative RT-PCR, a differential expression profile of invA during rickettsial host cell infection was examined. The invA transcript temporarily increased during the early period of infection. Expression of rickettsial groEL, a molecular indicator of cellular stresses, was also shown to be upregulated during the early period of infection. Furthermore, invA was cotranscribed in a polycistronic message with rrp, a gene encoding the response regulator protein homolog, which is a part of a two-component signal transduction system. These results support our earlier findings that under such stress conditions dinucleoside oligophosphate pyrophosphatase may function as a buffer, enhancing rickettsial survival within the cytoplasm of a eukaryotic cell. The expression of rickettsial dinucleoside oligophosphate pyrophosphatase may be regulated by a part of the two-component signal transduction system similar to that described for response regulators in other bacterial systems

Enhancing Epidemic Detection Using Syndromic Surveillance and Early Notification Methods

ObjectiveThis paper presents an investigation using early notification methods to enhancing epidemic detection in syndromic surveillance data from royal Thai army in Thailand.IntroductionEarly Notification Detection Systems have taken a critical role in providing early notice of disease outbreaks. To improve the detection methods for disease outbreaks, many detection methods have been created and implemented. However, there is limited information on the effectively of syndromic surveillance in Thailand. Knowing the performance, strengths and weakness of these surveillance systems in providing early warning for outbreaks will increase disease outbreak detection capacity in Thailand.MethodsThis study describes and compares the capabilities of various outbreak detection algorithms using 37,043 unique syndromic daily reports based on medical information from both civilian and military personnel from the Unit Base Surveillance of Royal Thai Army (RTA) along the Thai-Myanmar and Thai-Cambodia boarder areas. Traditional epidemic detection method: mean plus two SD were compared with algorithms for early notification methods and which included regression, regression/EWMA/Poisson, CDC-C1, CDC-C2 and CDC-C3. Early notification and epidemic detection methods were compared according to their ability to generate alert notifications. Sensitivity, specificity, positive predictive value (PPV), negative predictive value and overall accuracy to detect or predict disease outbreaks were estimated.ResultsThis study shows that the preliminary results are promising for epidemic detection by early notification methods in syndromic surveillance in Thailand. The majority of syndromic records were categorized into 12 symptoms. The three most common symptoms were respiratory, fever and gastrointestinal illness (11,501; 9,549 and 4,498 respectively). The results from the early notification systems were analyzed and their performances were compared with traditional epidemic detection method according to their ability to generate early warning alerts for the 3 symptoms. In our study regression/EWMA/Poisson method had higher specificity across the 3 symptoms (94.5%, 94.7% and 95.9% respectively), but generated lower sensitivity (22.6%, 40.4% and 23.1%). CDC-C1, CDC-C2 and CDC-C3 algorithms are easy to understand and are widely used. CDC-C3 had higher sensitivity to detect gradual disease outbreak effects (64.2%, 70.2% and 57.7%), but it is known to produce higher alarm rates/false positive signals.ConclusionsWithin the syndromic surveillance data of RTA, the CDC algorithm is the best chosen to use in the syndromic system due to being easy to understand and implement in a system with high sensitivity. CDC-C2 is the best early notification detection method due to its high sensitivity and PPV. However, CDC-C3 is shows the highest sensitivity, but exhibits the lowest specificity and PPV for all symptoms including a high alarm rates. To be useful, early notification detection methods must have acceptable operating characteristics. Consequently, we should select the most appropriate algorithm method to explain the data well and in order to improve detection of outbreaks. The comparison methods used in this study may be useful for testing other proposed alert threshold methods and may have further applications for other populations and other diseases.References1. Chretien JP, Burkom HS, Sedyaningsih ER, Larasati RP, et al. Syndromic Surveillance: Adapting Innovations to Developing Settings. PLoS Medicine 2008; vol 5: page 1-6.2. Burkom HS, Elbert Y, Magruder SF, Najmi AH, Peter W, Thompson MW. Developments in the roles, features, and evaluation of alerting algorithms for disease outbreak monitoring. Johns Hopkins APL Technical Digest 2008; vol 27: page 313

Development of multiplex PCR for neglected infectious diseases.

Scrub typhus, murine typhus, and leptospirosis are widely neglected infectious diseases caused by Orientia tsutsugamushi, Rickettsia typhi, and pathogenic Leptospira spp., respectively. Patients usually present with non-specific symptoms and therefore are commonly diagnosed with acute undifferentiated febrile illness. Consequently, patients face delayed treatment and increased mortality. Antibody-based serological test currently used as gold standard has limitations due to insufficient antibody titers, especially in the early phase of infection. In this study, we aimed to develop multiplex PCR to combine 3 primer pairs that target specific genes encoding 56-kDa TSA of O. tsutsugamushi, 17-kDa antigen of R. typhi, and LipL32 of L. Interrogans and evaluate its performance in comparison to the standard serological tests. Using EDTA blood samples of known patients, the sensitivity and specificity of our multiplex PCR was 100% and 70%, respectively. In addition, the assay was able to diagnose the co-infection of scrub typhus and leptospirosis. The assay may be useful in identifying causative agents during the early phase of these diseases, enabling prompt and appropriate treatment

Scrub Typhus Outbreak, Northern Thailand, 2006–2007

During a scrub typhus outbreak investigation in Thailand, 4 isolates of O. tsutsugamushi were obtained and established in culture. Phylogenetic analysis based on the 56-kDa type-specific antigen gene demonstrated that the isolates fell into 4 genetic clusters, 3 of which had been previously reported and 1 that represents a new genotype

Isolation and Propagation of the Ap-Variant 1 Strain of Anaplasma phagocytophilum in a Tick Cell Line▿

The first tissue culture isolates of the unique Anaplasma phagocytophilum strain, Ap-Variant 1, were obtained in the Ixodes scapularis tick-derived cell line ISE6. Two isolates were from goat blood samples: one from a goat infected with I. scapularis ticks from Rhode Island and a second from a goat infected by serial passage of blood from the first infected goat. Eight isolates were made directly from I. scapularis ticks collected from white-tailed deer in Minnesota and represent the first isolations of an Anaplasma species directly from ticks. Each of the 10 isolates had a 16S rRNA gene sequence identical to that previously described for Ap-Variant 1, but differences within the ank gene were found that suggest natural variation. Prevalence of Anaplasma in the Minnesota ticks was 63.9%; 23 of 36 ticks tested by PCR were positive. Six of the tick-derived isolates were obtained from a set of 18 PCR-positive ticks, for a 33.3% isolation success rate. The conservation of host tropism among the Rhode Island and Minnesota isolates of Ap-Variant 1 was examined by use of experimental infections of mice and a goat. A Minnesota tick-derived isolate (MN-61-2) was used to inoculate naïve animals, and this isolate was able to infect a goat but unable to infect each of five mice, confirming that the Minnesota isolates have the same host tropism as Ap-Variant 1 from the northeastern United States. Light and electron microscopy of the Ap-Variant 1 isolate MN-61-2 in ISE6 cells showed cytoplasmic inclusions characteristic of A. phagocytophilum with pleomorphic bacteria in membrane-bound vacuoles and both electron-dense and electron-lucent forms

Scrub Typhus Outbreak in Chonburi Province, Central Thailand, 2013

Investigation of a scrub typhus outbreak in Thailand during September 2013 found that 9.1% of Thai soldiers and 11.1% of residents living in areas surrounding training sites had antibodies against the causative agent, Orientia tsutsugamushi. Sequence analysis of O. tsutsugamushi from rodents and chiggers identified 7 genogroups and 3 genotypes