Todd E. Myers

Novel Bartonella Agent as Cause of Verruga Peruana

While studying chronic verruga peruana infections in Peru from 2003, we isolated a novel Bartonella agent, which we propose be named Candidatus Bartonella ancashi. This case reveals the inherent weakness of relying solely on clinical syndromes for diagnosis and underscores the need for a new diagnostic paradigm in developing settings.

Development of three quantitative real-time PCR assays for the detection of Rickettsia raoultii, Rickettsia slovaca, and Rickettsia aeschlimannii and their validation with ticks from the country of Georgia and the Republic of Azerbaijan.

A previous surveillance study of human pathogens within ticks collected in the country of Georgia showed a relatively high infection rate for Rickettsia raoultii, R. slovaca, and R. aeschlimannii. These 3 spotted fever group rickettsiae are human pathogens: R. raoultii and R. slovaca cause tick-borne lymphadenopathy (TIBOLA), and R. aeschlimannii causes an infection characterized by fever and maculopapular rash. Three quantitative real-time polymerase chain reaction (qPCR) assays, Rraoul, Rslov, and Raesch were developed and optimized to detect R. raoultii, R. slovaca, and R. aeschlimannii, respectively, by targeting fragments of the outer membrane protein B gene (ompB) using species-specific molecular beacon or TaqMan probes. The 3 qPCR assays showed 100% specificity when tested against a rickettsiae DNA panel (n=20) and a bacteria DNA panel (n=12). The limit of detection was found to be at least 3 copies per reaction for all assays. Validation of the assays using previously investigated tick nucleic acid preparations, which included Rickettsia-free tick samples, tick samples that contain R. raoultii, R. slovaca, R. aeschlimannii, and other Rickettsia spp., gave 100% sensitivity for all 3 qPCR assays. In addition, a total of 65 tick nucleic acid preparations (representing 259 individual ticks) collected from the country of Georgia and the Republic of Azerbaijan in 2009 was tested using the 3 qPCR assays. R. raoultii, R. slovaca, and R. aeschlimannii were not detected in any ticks (n=31) from the Republic of Azerbaijan, but in the ticks from the country of Georgia (n=228) the minimal infection rate for R. raoultii and R. slovaca in Dermacentor marginatus was 10% and 4%, respectively, and for R. aeschlimannii in Haemaphysalis sulcata and Hyalomma spp. it was 1.9% and 20%, respectively.

Detecting Rickettsia parkeri infection from eschar swab specimens.

The typical clinical presentation of several spotted fever group Rickettsia infections includes eschars. Clinical diagnosis of the condition is usually made by analysis of blood samples. We describe a more sensitive, noninvasive means of obtaining a sample for diagnosis by using an eschar swab specimen from patients infected with Rickettsia parkeri.

Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

ABSTRACT Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping.

Seroconversions to Rickettsiae in US Military Personnel in South Korea.

To the Editor: Infections with typhus group rickettsiae (TGR), spotted fever group rickettsiae (SFGR), and scrub typhus group orientiae (STGO) have been reported among persons in South Korea in increasing numbers over the past decade (1,2). During 2001–2011 in South Korea, 51,825 orientiae group infections were reported (mean incidence 9.95 cases/100,000 residents/year) (2). TGR (Rickettsia typhi), SFGR (R. akari, R. japonica, R. monacensis, and R. felis), and STGO (Orientia tsutsugamusi) have been identified in their arthropod vectors and reservoirs in northern provinces and at US military training facilities in South Korea (3–5). Currently, little data exist on the risk for rickettsioses and scrub typhus for US military deployed to South Korea. Thus, a retrospective serologic investigation to determine the level of exposure to rickettsiae among 9,303 military personnel deployed to South Korea was conducted. The study used de-identified predeployment and postdeployment serum samples made available from the Department of Defense Serum Repository (6). The study group consisted of men in combat-related jobs at US military training sites and military installations in South Korea during 1990–1995 while on active duty continuously for >1 year. This study protocol was reviewed and approved by the Naval Medical Research Command Institutional Review Board in compliance with all applicable federal regulations governing the protection of human subjects. Age range of the 9,303 soldiers in the study group was 17–52 (median 24) years. Most (99.6%) were stationed in Dongducheon, Yongtaeri, and Seoul, located in the Gyeonggi and Gangwon provinces in northern South Korea. Primary military occupation specialties were infantryman (58.8%), fighting vehicle infantryman (22.4%), indirect fire infantryman (12.3%), and heavy anti-armor weapons infantryman (6.5%). The soldiers’ postdeployment serum samples (n = 9,303) were screened at a dilution of 1:100 for IgG against TGR, SFGR, and STGO by using group-specific ELISA whole-cell antigen preparations from R. typhi Wilmington, R. conorii Morrocan, and a mixture of O. tsutsugamushi Karp, Kato, and Gilliam, respectively (7,8). TGR, SFGR, and STGO IgG ELISA titers (range 100–>6,400) were determined for screen-positive (net absorbance >0.500) postdeployment serum samples, and results were compared with matched predeployment serum samples. Samples with a net total absorbance >1.000 for serum dilutions 1:100, 1:400, 1:1,600, and 1:6,400 were considered titer positive. The inverse of the highest dilution of titer positive serum that produced a net absorbance >0.200 was determined to be the titer. Serum samples from laboratory animals infected with R. felis reacted specifically in the SFGR ELISA but not in the TGR ELISA (K.R. Macaluso and A.L. Richards, unpub. data); thus, any soldier infected with R. felis would have reacted in the SFGR but not the TGR ELISA. The postdeployment seropositivity in US military personnel for antibodies against TGR, SFGR, and STGO at a titer ≥100 were 1.3% (117/9,249), 9.0% (805/8,918), and 0.5% (44/9,135), respectively (Figure). Seropositivity occurred for 10 (0.1%), 181 (2.0%), and 15 (0.2%) men who showed evidence of infection (seroconversion or 4-fold rise in antibody titer) with TGR, SFGR, and STGO, respectively, during their deployment to South Korea (Figure). The chance of a soldier having an infection with SFGR was significantly higher than the chance of having an infection with TGR or STGO (χ2 test, p<0.05) (analysis performed in SAS version 9.4; SAS Institute Inc., Cary, NC, USA). For personnel who seroconverted or had a 4-fold rise in titer to TGR, SFGR, or STGO, the age range was 19–49 (median 25) years, and job specialties were infantrymen (63.5%), fighting vehicle infantrymen (16.4%), indirect fire infantrymen (14.2%), and heavy anti-armor weapons infantrymen (5.9%). Figure Evidence of rickettsiosis or scrub typhus among US military personnel deployed to South Korea. Black bars indicate postdeployment serum samples from US military personnel with a titer ≥1:100 (seropositive) to typhus group rickettsiae (TGR), spotted ... These results indicate that many US military personnel were exposed to rickettsiae and orientiae before their deployment to South Korea (Figure), perhaps because of previous deployments around the world or because of exposure to rickettsial agents at home (8–10). However, 206 (2.2%) of the men became infected with either a typhus group (n = 10) or spotted fever group (n = 181) rickettsia or a scrub typhus group orientia (n = 15) during their deployment to South Korea. More SFGR infections occurred than TGR and STGO infections, although the pathogens for the latter infections (R. typhi and O. tsutsugamushi) are considered endemic to South Korea and are believed to affect the public and military health more than SFGR (3). The SFGR infections might correlate with recent observations of highly prevalent rickettsia-infected tick and R. felis–infected flea populations seen in South Korea (4,5). No evidence of co-infection was found in the men assessed during the deployment. These results suggest a risk for rickettsial disease, including scrub typhus and especially spotted fever, among US military personnel stationed in or visiting South Korea.

Clinical, Serological, and Molecular Observations from a Case Series Study during the Asian Lineage Zika Virus Outbreak in Grenada during 2016

This paper describes the spatial and temporal distribution of cases, demographic characteristics of patients, and clinical manifestations of Zika virus (ZIKV) during the 2016 outbreak in Grenada. The first reported case was recorded in St. Andrew Parish in April, and the last reported case was seen in November, with peak transmission occurring in the last week of June, based on test results. Data were collected from a total of 514 patients, of whom 207 (40%) tested positive for ZIKV. No evidence was found that testing positive for ZIKV infection was related to age, gender, or pregnancy status. Clinical presentation with rash (OR = 2.4, 95% CI = 1.5 to 3.7) or with lymphadenopathy (OR = 1.7, 95% CI = 1.0 to 2.9) were the only reported symptoms consistent with testing positive for ZIKV infection. During the Zika outbreak, the infection rate was 20 clinical cases per 10,000 in the population compared to 41 cases per 10,000 during the chikungunya outbreak in Grenada in 2014 and 17 cases per 10,000 during the dengue outbreak in 2001-2002. Even though the country has employed vector control programs, with no apparent decrease in infection rates, it appears that new abatement approaches are needed to minimize morbidity in future arbovirus outbreaks.

Seroconversions for Coxiella and Rickettsial Pathogens among US Marines Deployed to Afghanistan, 2001-2010.

We assessed serum samples from 1,000 US Marines deployed to Afghanistan during 2001–2010 to find evidence of 4 rickettsial pathogens. Analysis of predeployment and postdeployment samples showed that 3.4% and 0.5% of the Marines seroconverted for the causative agents of Q fever and spotted fever group rickettsiosis, respectively.