Murine typhus and febrile illness, Nepal.

Murine typhus was diagnosed by PCR in 50 (7%) of 756 adults with febrile illness seeking treatment at Patan Hospital in Kathmandu, Nepal. Of patients with murine typhus, 64% were women, 86% were residents of Kathmandu, and 90% were unwell during the winter. No characteristics clearly distinguished typhus patients from those with blood culture-positive enteric fever.

Validation of a Rickettsia prowazekii-specific quantitative real-time PCR cassette and DNA extraction protocols using experimentally infected lice.

The presence of R. prowazekii in lice using R. prowazekii-specific qPCR cassettes showed the changes of the rickettsial burden in the lice post-infection.

Clinic-based surveillance for bacterial- and rotavirus-associated diarrhea in Egyptian children.

To identify enteropathogens for vaccine development, we implemented clinic-based surveillance for severe pediatric diarrhea in Egypt's Nile River Delta. Over 2 years, a physician clinically evaluated and obtained stool samples for microbiology from patients with diarrhea and less than 6 years of age. In the first (N = 714) and second clinic (N = 561), respectively, 36% (N = 254) and 46% (N = 260) of children were infected with rotavirus, enterotoxigenic Escherichia coli (ETEC), Campylobacter, or Shigella. When excluding mixed rotavirus-bacterial infections, for the first and second clinic, 23% and 10% had rotavirus-associated diarrhea, and 14% and 17% had ETEC-associated diarrhea, respectively. Campylobacter-associated diarrhea was 1% and 3%, and Shigella-associated diarrhea was 2% and 1%, respectively, for the two clinics. Rotavirus-associated diarrhea peaked in late summer to early winter, while bacterial agents were prevalent during summer. Rotavirus-associated cases presented with dehydration, vomiting, and were often hospitalized. Children with Shigella- or Campylobacter-associated diarrhea reported as watery diarrhea and rarely dysentery. ETEC did not have any clinically distinct characteristics. For vaccine development and/or deployment, our study suggests that rotavirus is of principle concern, followed by ETEC, Shigella, and Campylobacter.

Enterotoxigenic Escherichia coli colonization factor types collected from 1997 to 2001 in US military personnel during operation Bright Star in northern Egypt.

Operation Bright Star (OBS) is a biennial, multinational exercise in Egypt involving 15000 US troops. Consistent with past observations in deployed troops, diarrhea is the most significant cause of morbidity. Focused efforts are ongoing to develop vaccines against the most common pathogens affecting our troops. As part of these efforts, diarrhea surveillance was conducted during OBS to monitor pathogens associated with illness and to identify new vaccine targets. A retrospective review was conducted of prior studies with similar methods. Soldiers with diarrhea presenting to the OBS clinic provided a stool sample that was inoculated into Carey-Blair transport media. Within 3 days, the Cary-Blair tubes were transported to the Naval Medical Research Unit no. 3 in Cairo where bacterial culture was performed. As part of the evaluation, 5 Escherichia coli-like colonies were collected and tested for toxin production using the GM1-ELISA. Toxin-positive isolates were further tested for colonization factors (CF) by a dot-blot assay using a standardized panel of monoclonal antibodies against CFA/I, CS1-CS7, CS17, CS8 (CFA/III), CS12 (PCFO159), and CS14 (PCFO166). Enterotoxigenic E. coli (ETEC) was the most frequently isolated pathogen during each OBS from which data were collected. The rate of ETEC-associated diarrhea ranged from 22% to 58%. Over time, there were dramatic shifts in the frequency and distribution of CFs. Over the 5 years of study, an increasing number of ETEC isolates had no known CF identified, and in 2001, only 40% of ETEC was associated with known CFs. The most commonly identified CF was CS6. Diarrheal disease, particularly ETEC, continues to be a common malady among US military personnel deployed to Egypt. We have identified ETEC CF types, especially CS6, which should be considered potential vaccine candidates. However, despite intensive testing, CFs could not be identified in most of the ETEC isolated, highlighting the need for further studies to identify novel CFs and alternative vaccine targets.

Prevalence, Distribution, and Development of an Ecological Niche Model of Dermacentor variabilis Ticks Positive for Rickettsia montanensis.

Rickettsia montanensis has long been considered a nonpathogenic member of the spotted fever group rickettsiae. However, the infection potential of R. montanensis is being revisited in light of its recent association with a case of human infection in the United States and the possibility that additional cases may have been misdiagnosed as Rocky Mountain spotted fever. To this end, DNA was extracted from American dog ticks (Dermacentor variabilis) removed from Department of Defense (DoD) personnel and their dependents at DoD medical treatment facilities (MTFs) during 2002-2012 (n = 4792). These 4792 samples were analyzed for the presence of R. montanensis (n = 36; 2.84%) and all vector DNA was confirmed to be of D. variabilis origin using a novel Dermacentor genus-specific quantitative real-time polymerase chain reaction procedure, Derm, and a novel Dermacentor species multilocus sequence typing assay. To assess the risk of R. montanensis infection, the positive and negative samples were geographically mapped utilizing MTF site locations. Tick localities were imported into a geographical information systems (GIS) program, ArcGIS, for mapping and analysis. The ecological niche modeling (ENM) program, Maxent, was used to estimate the probability of tick presence in eastern United States using locations of both R. montanensis-positive and -negative ticks, climate, and elevation data. The ENM for R. montanensis-positive D. variabilis estimated high probabilities of the positive ticks occurring in two main areas, including the northern Midwest and mid-Atlantic portions of the northeastern regions of United States, whereas the R. montanensis-negative D. variabilis tick model showed a wider estimated range. The results suggest that R. montanensis-positive and -negative D. variabilis have different ranges where humans may be at risk and are influenced by similar and different factors.

Diarrheal illness among deployed U.S. military personnel during Operation Bright Star 2001--Egypt.

In the fall of 2001, approximately 15,000 U.S. military personnel participated in a military exercise in the northwestern Egyptian desert. To assess the prevalence and impact of diarrhea and enteropathogen distribution, we conducted a post-deployment survey and a case series study. A departure convenience sampling (n = 3725) was used in the post-deployment survey. Overall, 9.3% reported diarrhea, 2.6% sought medical care, and 2.8% stopped or decreased their work for at least a day. Among those reporting diarrhea, 41.7% had symptoms for less than 2 days, 43.5% had symptoms from 2-5 days, and 14.8% had symptoms for more than 5 days. In the case series study, pathogens were identified in 53.6% of the 129 cases enrolled. Pathogens identified included enterotoxigenic E. coli (n = 53), enteroaggregative E. coli (n = 13), Cryptosporidium (n = 9), Campylobacter jejuni (n = 7), noroviruses (n = 7), Shigella flexneri (n = 2), rotavirus (n = 2), and Entamoeba histolytica (n = 2). Among those seeking care for diarrhea, two thirds reported a decreased ability or inability to perform their jobs for at least one day, but overall, diarrhea was much less prevalent than in past surveys in this region, with minimal impact on the mission.

The epidemiology of travelers' diarrhea in Incirlik, Turkey: a region with a predominance of heat-stabile toxin producing enterotoxigenic Escherichia coli.

This study evaluated travelers' diarrhea among US military personnel on short-term deployment to Incirlik Air Base, Turkey, from June through September 2002. Upon reporting for care for travelers' diarrhea, subjects were enrolled into the study and completed a series of questionnaires and provided stool specimens for pathogen identification and antimicrobial susceptibility testing. Fifty-three percent of the 202 participating subjects had a pathogen isolated from their stool. Enterotoxigenic Escherichia coli (ETEC) was the predominant pathogen (41%), followed by Campylobacter spp. (12%). The most common ETEC phenotype recovered was stable toxin (ST) CS6 (47% of all ETEC). Most (91.1%) of the cases presented with water diarrhea regardless of isolated pathogen. However, there were some differences in nongastrointestinal symptoms among subjects with Campylobacter spp. All illnesses were well managed with antibiotics with or without loperamide with a median time to the last unformed stool of 9 h (interquartile range, 1-32 h). We found no food or environmental factors associated with a differential risk of infection with a specific pathogen. Travelers' diarrhea among a US military population in and around Incirlik, Turkey, can commonly be attributed to ETEC and Campylobacter spp. The high proportion of ST-only-producing CS6 ETEC in this region highlights the pathogen's worldwide diversity. Future studies of travelers' diarrhea in this population should adapt more novel microbiologic techniques such as polymerase chain reaction and enhanced culture methods to increase the likelihood of identifying pathogenic E. coli.

Development of quantitative real-time PCR assays to detect Rickettsia typhi and Rickettsia felis, the causative agents of murine typhus and flea-borne spotted fever.

Rickettsia typhi and Rickettsia felis are the etiologic agents of murine typhus and flea-borne spotted fever, respectively. We have constructed two quantitative real-time polymerase chain reaction (qPCR) assays to detect these pathogenic rickettsiae. The qPCR assays were developed utilizing unique sequences of the R. typhi and R. felis outer membrane protein B genes (ompB) to design the specific primers and molecular beacon probes. The assays were found to be species-specific and did not yield false-positive reactions with nucleic acid from other rickettsiae, orientiae, neorickettsiae or unrelated bacteria. In addition, the assays were sensitive enough to detect three target sequence copies per reaction and were capable of detecting R. typhi and R. felis nucleic acid in the cat flea, Ctenocephalides felis. These results demonstrate that two sensitive and specific qPCR assays have been successfully developed to detect and enumerate R. typhi and R. felis.

Sensitivity and specificity of various tests for the diagnosis of Helicobacter pylori in Egyptian children.

OBJECTIVES: Many noninvasive methods (using breath, blood, and stool samples) are available to diagnose Helicobacter pylori. However, because the noninvasive tests are proxy measures of the infection, they need validation before use. Factors that may affect test validity include patient age, gender, and geographic location. Because no data were available on the validation of noninvasive tests for the diagnosis of H. pylori among children in the Middle East, this study was performed. METHODS: Children between 2 and 17 years of age evaluated at the Cairo University School of Medicine pediatric gastroenterology clinic who were already scheduled for upper endoscopy were eligible for enrollment in the study. At the time of endoscopy, 3 biopsies were collected and used for rapid urease, histology, and culture, respectively. All children also donated a sample of stool and blood and had a urea breath test performed. Stool and serum samples were tested for the presence of H. pylori by using commercially available enzyme-linked immunosorbent assay-based technology. The sensitivity, specificity, and positive and negative predictive values were calculated for each noninvasive test used in the study. Receiver operating curves also were charted to determine optimal cut points for the various tests when used in the current study cohort. RESULTS: One hundred eight children were enrolled in the study, with 52 children being under 6 years of age. The urea breath test and HpStar (DakoCytomation, Norden, Denmark) stool enzyme-linked immunosorbent assay kit had the highest sensitivity and specificity (sensitivity and specificity: 98 and 89 [urea breath test] and 94 and 81 [HpStar], respectively), whereas the serologic kit had an unacceptably low sensitivity (50%). The sensitivity of neither the urea breath test nor the HpStar tests was affected by subject age, but specificity of the HpStar test, although still high, was significantly lower among children under 6 years. Receiver operating curves found optimal cut points of the urea breath test at 6.2 delta over baseline and of the HpStar at 0.25 enzyme-linked immunosorbent assay units. CONCLUSION: The urea breath test and HpSTAR stool antigen kit are reliable tests for the noninvasive diagnosis of H. pylori among children living in the Middle East.

Posttreatment changes in Escherichia coli antimicrobial susceptibility rates among diarrheic patients treated with ciprofloxacin.

Changes in antimicrobial resistance of Escherichia coli among deployed U.S. military personnel being treated for diarrhea were evaluated. Stool samples were collected pretreatment and on days 7, 14, and 28 posttreatment. Resistance to ciprofloxacin was noted in 13.3% of baseline specimens, and rates of resistance against multiple antibiotics increased dramatically from baseline to day 7 and then tapered off to return to pretreatment levels by day 28, except for ciprofloxacin, suggesting that population accumulative usage of fluoroquinolones may result in an incremental increase in resistance rates.

Human Infection with Rickettsia honei, Thailand.

Human spotted fever rickettsiosis was detected molecularly by 2 real-time polymerase chain reaction (PCR) assays performed on DNA extracted from a Thai patient's serum sample. Sequences of PCR amplicons from 5 rickettsial genes used for multilocus sequence typing were 100% identical with those deposited with GenBank for Rickettsia honei TT-118.

Universal high-level primary metronidazole resistance in Helicobacter pylori isolated from children in Egypt.

Antimicrobial susceptibility testing was performed on 48 isolates of Helicobacter pylori recovered from Egyptian children undergoing routine endoscopies. The isolates were universally highly resistant to metronidazole, but resistance to other tested antimicrobial agents was rare (4% for clarithromycin, erythromycin, and azithromycin resistance versus 2% for ciprofloxacin and ampicillin resistance). Use of metronidazole for the treatment of H. pylori in Egypt should be avoided.