Browsing by Author "Gray, Gregory C"

Introduction: Extensive studies on tickborne pathogens (TBPs) have been conducted in developed nations, relatively less has been done in developing nations leaving a large gap of knowledge. Mongolia, a country built upon nomadic culture and pastoralism is an ideal system to study TBPs as the population is at an increased risk due to increased time spent outside herding livestock. Discoveries of TBPs in Mongolia include Babesia spp., Anaplasma spp., Borrelia spp., Rickettsia spp. and tick-borne encephalitis virus. While research has focused on TBPs in humans and ticks in Mongolia, little research has assessed animal reservoirs, specifically small mammal species, as reservoirs for TBPs. This project aimed to 1) identify the role of small mammal species in the ecology of TBPs in Mongolia, specifically Rickettsia spp., Anaplasma spp., and Borrelia spp. using serological and molecular analysis and 2) identify risk factors associated with the prevalence of TBPs in small mammal populations in Mongolia.

Methods: From June to July 2016, rodents were live-trapped, and whole blood, serum and ear biopsy samples were collected. Sixty-four rodents were trapped in three aimags (provinces) in northern Mongolia. Whole blood samples were tested by PCR to detect the presence of Rickettsia spp., Anaplasma spp., and Borrelia spp.. In addition, ear biopsy samples were tested by PCR to detect the presence of Borrelia spp.. All rodents were serologically tested for antibodies to Anaplasma phagocytophilum and Rickettsia rickettsii. A multivariate model was used to assess risk factors for the presence of tickborne pathogens. Risk factors examined included species and sex of animal, location and presence of ticks.

Results: 56.0%, 39.0% and 0.0% of animals were positive by PCR for Borrelia spp., Rickettsia spp. and Anaplasma spp., respectively. 41.9% and 24.2% of animals were seropositive for A. phagocytophilum and Rickettsia rickettsii, respectively. Risk factors found to be important predictors of Borrelia spp. molecular detection included small mammal capture in Tov aimag (OR, 4.1; 95% CI, 1.00 – 16.80), male small mammals (OR, 3.07; 95% CI, 0.99 – 9.51) and ground squirrel species type (OR, 3.24; 95% CI, 0.90 – 11.70). The risk factor found to be an important predictor of Rickettsia spp. molecular detection was Mongolian gerbil species type (OR, 246.5; 95% CI, 20.77 – 2925.88). Presence of ticks on small mammals (OR, 4.62; 95% CI, 0.92 – 23.24) was an important risk factor for A. phagocytophilum antibody detection. No risk factors were identified as being important predictors of antibody detection of R. Rickettsii.

Conclusion: The results of this study provide considerable evidence of TBPs circulating in small mammal populations in Northern Mongolia. Further information on TBPs in ticks, humans, livestock and wildlife reservoirs will be important to address public health interventions for TBPs in Mongolia in the future.

Background

Melioidosis is a neglected tropical disease that is highly prevalent in Southeast Asia. Misdiagnoses are common as the presenting symptoms are similar to other diseases including upper respiratory infections. When not treated with antibiotics, the disease can lead to severe morbidity or death. Current diagnostics in low- and middle-income countries are often not sensitive nor rapid. Point-of-care rapid diagnostic tests (POC-RDTs) are a potential solution. Few studies have compared the accuracy of POC-RDTs and molecular assays against blood culture. The goal of this study was to conduct such comparisons in detecting Burkholderia pseudomallei infections among infection-suspected patients in Kapit, Sarawak, Malaysia.

Methods

We used an informed consent process as approved by two institutional review boards. In this cross-sectional study, we engaged patients meeting a melioidosis-like case definition that included classical symptoms such as prolonged fever with joint pain and/or abscess. We studied the patients routinely collected clinical specimens with a POC-RDT (Active Melioidosis DetectTM) and a molecular assay compared with the B. pseudomallei bacterial culture for isolation of the bacterial organism.

Results

One hundred patients aged 6 months - 79 years from Kapit Hospital were enrolled in the study from June 12, 2018 to January 8, 2019. Of the 100 sera, 97 urine, and 16 bodily fluid samples (total n= 213) tested with the RDT, 23 samples gave positive results (7 sera, 15 urine, and 1 bodily fluids). Compared to the molecular assay, the POC-RDT had a sensitivity of 40% (95% CI, 5%- 85%), specificity of 94% (95% CI, 87% - 98%), and an accuracy of 90% (95 CI, 82% - 95%) for sera; and a sensitivity of 80% (95% CI, 28%- 99%),a specificity of 65% (95% CI, 55% - 75%), and an accuracy of 87% (95 CI, 77% - 94%) for urine; and a sensitivity of 80% (95% CI, 28%- 99%), a specificity of 65% (95% CI, 55% - 75%), and an accuracy of 81% (95 CI, 54% - 96%) for other bodily fluids. Additionally, when compared to the bacterial culture results, the POC-RDT showed a sensitivity of 38% (95% CI, 9%- 76%), specificity of 95% (95% CI, 88% - 99%), and an accuracy of 90% (95 CI, 82% - 95%) for sera; a sensitivity of 88% (95% CI, 47%- 100%), a specificity of 88% (95% CI, 77% - 95%), and an accuracy of 94% (95 CI, 84% - 98%) for urine; and a sensitivity of 25% (95% CI, 1%- 81%), a specificity of 100% (95% CI, 74% - 100%), and an accuracy of 81% (95 CI, 54% - 96%) for other bodily fluids.

Conclusion

While study enrollment will continue, data from the first 100 participants, suggests the POC-RDT had poor sensitivity, good accuracy, and high specificity in detecting B. pseudomallei infection. Thus far, the POC-RDT assay seems to work better on urine specimens. Due to low sensitivity, the study data do not support recommending POC-RDT strips as a single diagnostic method. However, as the POC-RDT had high specificity when the test is positive it seems appropriate for clinicians to assume the patient is infected and to prescribe specific antimicrobial therapy. While more participant data are needed, it seems likely that the POC-RDT could be useful in helping physicians to begin treatment early with the high specificity that the POC-RDT has exhibited. If paired with an RDT with high sensitivity, this POC-RDT would add a great value to infection management.

Background: Recent epidemiological studies demonstrate that the prevalence of the fifth major human malaria parasite, Plasmodium knowlesi (monkey malaria), is often underestimated and misdiagnosed with standard microscopy blood film. We sought to adapt and compare a new simple molecular diagnostic method for P. knowlesi with the gold standard nested molecular assay and microscopy blood film in P. knowlesi hotspot areas in Sarawak, Malaysia. In addition, we analyzed the statistical association between P. knowlesi positive test results and demographic and behavioral/occupational risk factors.

Methods: The study was conducted at Sibu, Kapit and Sarikei Hospitals in Sarawak, Malaysia. 115 blood samples were collected from malaria suspected patients seeking treatment at these hospitals. Samples were analyzed by microscopy, Nested polymerase chain reaction (PCR) and single-step PCR. Sensitivity, specificity, and practical value of the new single-step PCR assay was calculated. Bivariate and multivariate regression was conducted to test the possible risk factors for the detection of P. knowlesi.

Results: Single-step PCR showed low sensitivity (51.92%, 95%CI 37.63 - 65.99%) compared to nested PCR and 46.03% (95%CI 33.39 - 59.06%) compared to microscopy. When compared to nested PCR, microscopy had a false positive rate of 20.6%. However, it only missed 2 cases of P. knowlesi. The mean age in the study population was 40.35. Patients enrolled at Kapit hospital had higher odds ratio for positive P. knowlesi PCR results (adjusted OR = 4.46, 95%CI 1.16 – 11.51). Age above 21 years (adjusted OR = 6.28, 95%CI 1.53 – 25.64), male gender (adjusted OR = 2.46, 95%CI 0.91 – 6.65) and living near a vegetation (Plantation, forest, fruit trees or wet rice paddy) (adjusted OR = 5.96, 95%CI 1.11 – 31.83) were associated with increased risk for P. knowlesi infection.

Conclusions: Data from this study showed that single-step PCR has a low sensitivity and thus, it is not a suitable alternative for accurate detection of P. knowlesi. Further studies are required for assessment and development of other diagnostic assays or new primer sets. Multivariate analysis revealed that adult men over the age of 21 who live near agricultural areas have the highest risk for P. knowlesi malaria infection. Large- scale descriptive studies of both non-human hosts and vectors would greatly influence prevention and control strategies of this zoonotic disease.

Background: In recent years, emerging zoonotic microbes are gaining more attention from the public and policy makers. Explosive outbreaks such as those from avian influenza viruses, severe acute respiratory syndrome (SARS) virus, swine influenza viruses, Hendra virus, Nipah virus, and Middle East respiratory syndrome (MERS) have had tremendous international economic and social impact. In particular, livestock workers have been found to be at increased infection risk and some of the first impacted by a novel pathogen. One of the main obstacles in averting outbreaks of novel microbes is detecting it when it first begins to cross species from animals to man and may not cause severe disease. Often routine diagnostics will fail to detect a new pathogen. The purpose of this research was to evaluate diagnostics for emerging coronaviruses that would be missed with routine diagnostics.

Methods: In 2016, I learned how to run new diagnostics adapted at Duke University to detect novel coronaviruses. I took this molecular technology to Sarawak, Malaysia, where I applied the assays against a panel of human clinical specimens from patients seen at three hospitals for respiratory illnesses. Our collaborators in Sarawak had previously examined these specimens with other assays against human coronaviruses but did not tell me of their results.

Results: In my hands, the new pan-species coronavirus assay detected only one coronavirus among 88 clinical specimens. After I finished my assay work, I learned from our collaborators that 27of the 88 specimens had been positive for at least one previously recognized human coronavirus. Hence, the sensitivity of the new assay in my hands found to be 3.70% (95% confidence interval 0% - 11.91%). However, the assay accurately showed negative results with a specificity of 100%

Conclusion: While this low sensitivity may have been real, it may also been influenced by a number of confounding factors such as specimen nucleic acid degradation with numerous freeze-thaw cycles, imprecise adaptation of an assay to new equipment in a new laboratory, or my or our collaborators’ operator error. It is difficult to precisely identify the cause of the discordance. Nevertheless, I learned a great deal about global health in conducting this research in Sarawak and have chronicled some of these lessons in this report.

Introduction: According to the World Health Organization (WHO), 500 million arbovirus cases are diagnosed around the world annually, with 2.7 million associated deaths [1]. The burden of disease caused by dengue, chikungunya, and Zika viruses is likely to be underestimated due to a lack of accurate diagnostic tools and knowledge gaps regarding their epidemiology [2, 3]. This thesis uses a subset of data from an on-going 24-month study to evaluate the potential etiology of dengue-like symptoms of patients recruited from medical facilities in Sarawak, Malaysia. A secondary aim is to assess the diagnostic clinical effectiveness of a new detection method, the novel T-Cor 8 Multiplexing Platform (Tetracore, Inc., USA), using qRT-PCR assays as the gold standard method for comparison. The prevalence of arboviral infections as determined by gold-standard qRT-PCR assays and potential risk factors in the study population were also analysed.

Methods: In this cross-sectional study, patients more than seven years of age with dengue-like symptoms were enrolled at medical facilities in the towns of Sibu and Kapit in Sarawak, Malaysia. Blood, urine, and gingival crevicular fluid samples, as well as risk factor data, were collected from participants at the time of enrolment. These samples were studied by qRT-PCR assays and the novel T-Cor 8 Multiplexing Platform.

Results: Seven (14%) of 51 participants’ serum RNA samples tested positive for arbovirus infection by gold-standard qRT-PCR assays. Two participants (4%) were positive for dengue subtype-1, four participants (8%) were positive for dengue subtype-2, and one participant (2%) was positive for dengue subtype-4. No patient samples had molecular evidence of chikungunya or Zika viruses. The T-Cor 8 multiplexing platform demonstrated a 71% sensitivity (95% confidence interval 29-96%), 93% specificity (95% confidence interval 81-99%), and 90% accuracy (95% confidence interval 78-97%) compared to the gold-standard assays on serum RNA samples. From this subset of data, we failed to identify important risk factors for arboviral infection.

Conclusion: From this limited subset of data, we conclude that the T-Cor 8 platform’s simplicity and accuracy in detecting at least dengue virus infections has considerable potential for clinical usefulness in low-resource settings.

Introduction: Globally, acute respiratory infections (ARIs) are the leading cause of mortality and death among children below 5 years of age and adults of all ages. The disease burden due to ARIs is particularly high in low-to-middle income countries (LMICs) like Indonesia where the tropical climate, poverty-associated malnutrition, overcrowding, and air pollution increase the risk of infections from airborne pathogens. Although, the global burden of respiratory viruses such as respiratory syncytial virus, influenza, and enteroviruses has been broadly studied, there remains a lack of effective and continuous surveillance data to understand the full impact of respiratory viruses in Indonesia and other LMICs. Additionally, due to crowded conditions, inadequate ventilation, and poor sanitation, health care centers in LMICs may be hot spots for rapid transmission and exchange of respiratory viruses. In such settings, bioaerosol sampling, which is a low cost, rapid and noninvasive method for surveillance of respiratory pathogens, has been shown to be an effective tool for surveillance of respiratory viruses. Therefore, in this study, we conducted surveillance for influenza A virus and enterovirus using bioaerosol and surface swab sampling in a clinical setting located in Jakarta, Indonesia.

Methods: In this study, we performed bioaerosol sampling and surface swab sampling for 8 days during August 2019, inside a tertiary care hospital located in Eastern Jakarta, Indonesia. The sampling sites included a common waiting room, a triage room, and a physician’s room with clinical staff and patients present during sample collection. We used a SKC BioSampler with a BioLite Air Sampling Pump (run for 60 min at 5 L/min) with polytetrafluoroethylene Teflon filter cassettes and sterile nylon-flocked swabs to take surface swab samples. Temperature and humidity data were also collected at each sampling site concurrently. Viral RNA was extracted from bioaerosol samples and surface swabs using the QIAamp Viral RNA Mini Kit. Extracted Viral RNA was assessed with real-time RT-PCR (qRT-PCR) assays using the SuperScript® III Platinum One-Step qRT-PCR System with Platinum® Taq DNA Polymerase for the detection of influenza A and human enterovirus.

Results: Overall, 5 (7.8%) of the 64 specimens indicated evidence of Influenza A, with 4 (12.5%) positive for influenza A virus among 32 bioaerosol samples, and 1 (3.12%) positive for influenza A virus, among 32 surface swab samples. We repot no enteorvirus positive from our samples.

Conclusions: The gaps in surveillance of respiratory viruses from LMICs countries like Indonesia can be fulfilled by adopting bioaerosol sampling method, which is noninvasive, effective, cost friendly and rapid surveillance method. Combined with molecular assays, aerosol sampling has potential to detect respiratory viruses in health care settings. These surveillance techniques using bioaerosol samplings in one way, will help to generate evidence based scientific studies to identify prevalence of respiratory viruses in different settings, while in another way might facilitates to improve global and national polices for surveillance and early detection of respiratory viruses.

Abstract

Introduction: Mongolia is a country known for its rich nomadic and pastoral culture, with populations of people who work in environments that are densely populated with ticks and TBP animal reservoirs. TBPs typically undergo transstadial transmission, but transovarial transmission may also occur. Transovarial transmission events have been demonstrated in laboratory settings, but few studies have evaluated transovarial transmission of TBPs in field settings within the host-vector ecosystem. Tick borne pathogens of most concern in Mongolia are Rickettsia spp., Anaplasma spp., Borrelia spp., Babesia spp., Ehrlichia spp., and tick-borne encephalitis. In this study, specific aims were: 1) To determine the prevalence of tick borne pathogens, particularly Rickettsia spp., Anaplasma spp., and Ehrlichia spp. among various tick species at different developmental life stages; and 2) To understand the role of animal reservoirs and vertical transmission of TBPs among feeding ticks at different life stages using larval and nymph ticks collected in the wild from small mammal reservoirs, as well as eggs laid by engorged wild-caught adult female ticks and reared in the laboratory.

Methods: In this cross-sectional study, ticks in their larvae and nymph life stages were collected off of captured rodents across seven soums (districts) in three aimags (provinces) situated in the Northern region of Mongolia from June 20th to July 23rd, 2016. Engorged adult ticks were collected from livestock located in three soums within three aimags from May 6th to 22nd, 2016. Tick eggs were collected from engorged ticks from May 9th to June 1st, 2016. Ticks were tested by PCR to detect the presence of Rickettsia spp., Anaplasma spp., and Ehrlichia spp.

Results: There were 546 (88%) larval and 72 (12%) nymphal Dermacentor spp. ticks collected. There were 588 (95%) of 618 ticks allocated into 42 larvae and 18 nymph pools (60 pools total). All tick pools were PCR-positive for Rickettsia spp. and no tick pools were PCR-positive for Anaplasma/Ehrlichia spp. minimum infection rate (MIR) for R. raoultii ranged from 6.7% to 28.6%. Of the 60 tick pools, 50 (522 ticks total) were matched with rodent rickettsial infection history status. There were 31 (62%) tick pools or 362 (69%) of individual ticks found on rodents with no history of rickettsial infection. The majority of ticks discovered to have no association with rodents with rickettsial infection history were larvae (352/362 individual ticks). There were 38 adult fed female ticks collected. All adult fed ticks were PCR-positive for Rickettsia spp. and 2 (5%) were PCR-positive for Anaplasma/Ehrlichia spp. There were 33 ticks that laid eggs. PCR testing of eggs showed a 91% (30/33) positivity for Rickettsia spp. and one pool of eggs was PCR-positive for Anaplasma/Ehrlichia spp. All sequenced Rickettsia spp. products were identified to be R. raoultii and all sequenced Anaplasma/Ehrlichia spp. were An. ovis.

Conclusions: This study identified transovarial transmission of Rickettsia spp. and Anaplasma spp. among D. nuttalli ticks. This study also found a low association between rodents with history of Rickettsia spp. infection and infection status of biting ticks. Additional study is needed to further assess the proportion of transovarial transmission found in nature. Specifically, testing of individual tick eggs and larvae should be conducted. A better understanding of the ecology of TBPs in nature can provide public health and human and veterinary medicine with a greater awareness of the burden of TBPs in Mongolia.

Diarrheal diseases continue to be one of the most significant killers of children throughout the world, and many diarrheal diseases are zoonotic in nature. In Malaysia, not much is known about the viruses causing disease in humans and animals, and there is little evidence describing the role noroviruses play in diarrheal disease in the state of Sarawak. This study aimed to estimate the prevalence of noroviruses in children admitted to Sibu Hospital with acute diarrhea and the prevalence of norovirus in swine environments in Sarawak. We collected stool samples from children admitted to the hospital for acute gastroenteritis and by convenience sampling from registered pig farms. Stool samples were tested for norovirus genogroups I and II/IV. At the time of sample collection, information was collected about prior animal exposure and medical history including previous hospitalization for diarrhea. Of the 70 participants enrolled in the study, 3 tested positive for norovirus G.II/IV. None of the swine stool samples tested positive for noroviruses. None of the animal exposure variables were statistically associated with increased odds of previous hospitalization for diarrhea, but prior cat exposure non-significantly increased the odds of previous hospitalization by 3.78 (95% CI 0.89, 16.11). Although norovirus is not highly prevalent in children in Sibu, Malaysia, diarrheal disease causes significant disease burden in the study population. Future work should aim to elucidate risk factors for severe diarrhea and to determine the prevalence of other disease-causing pathogens. This information will help clinicians better treat their patients and public health officials design programs to minimize the risk exposure to prevent diarrheal disease.

Introduction: Pneumonia, despite its stereotype as a routine disease, remains the leading cause of morbidity and mortality among children under five worldwide, responsible for nearly 16% of all childhood deaths(1). With an imprecise definition and multiple etiologies, diagnosis and treatment of the disease is difficult when based solely on clinical and symptomatic manifestations(2). This study was conducted as a subset of an ongoing year-long study aimed to determine the viral etiology of and risk factors for pneumonia among 600 patients admitted to Sibu and Kapit Hospitals in Sarawak, Malaysia. Specifically, this sub-study examined molecular diagnostics for two common respiratory pathogens, which often infect children seen at these hospitals and which lacked any such diagnostic capability. We sought to determine the prevalence of respiratory syncytial virus (RSV) subtypes A and B and parainfluenza virus (PIV) types 1- 4. The study describes demographic, viral and behavioral risk factors for these admissions. Additionally, the study aimed to assess viral transmission in the air in hospital wards.

Methods: To determine the viral etiology of pneumonia cases, this cross-sectional study enrolled 129 patients over the age of one month, who had been diagnosed and hospitalized with pneumonia at Sibu or Kapit Hospital in Sarawak, Malaysia between June 15 and July 27, 2017. Nasopharyngeal (NP) swabs were collected and analyzed using real-time reverse transcription polymerase chain reaction (qRT-PCR) at Sibu Hospital’s Clinical Research Centre laboratory. A multivariable model was used to assess risk factors for the presence of different respiratory viruses.

Results: Of 129 specimens collected, 40 samples tested positive for RSV-A (31.01%), two were positive for RSV B (01.55%), one was positive for PIV-3 (0.78%) and one was positive for PIV-4 (0.78%). No samples were positive for PIV-1 or PIV-2. The prevalence of RSV-A was 46% (23/50) at Kapit Hospital and 21.52% at Sibu Hospital (17/79). In Sibu Hospital’s pediatric wards, one bioaerosol tested positive for adenovirus and two tested as suspect-positives for adenovirus. One (1) bioaerosol sample from an adult ward at Sibu Hospital tested as a suspect-positive for RSV-A. A multivariable analysis found risk factors of age (>1 year and 1-5 years vs > 5 years) and location of hospitalization (Kapit vs Sibu) potentially important predictors of RSV-A molecular detection.

Conclusions: During this brief demonstration study, we found a high prevalence of RSV-A among pneumonia patients admitted to the two hospitals. Having routine diagnostic capability for these viruses, particularly RSV, could help clinicians prescribe antiviral therapies which could reduce RSV morbidity and mortality.

Introduction: An estimated 75% of emerging infections in humans are zoonotic, posing a serious risk of future pandemics. The large livestock operations and dense human population of Southeast Asia are considered a hot-spot for the generation of novel viruses. The primary objective of this pilot study is to employ novel molecular laboratory analyses to examine evidence that swine pathogens including porcine circovirus 2, porcine rotaviruses, encephalomyocarditis virus, and porcine reproductive and respiratory syndrome virus, may be aerosolized at the animal-interface and that humans working in these environments may be carrying these viruses in their nasal airways.

Methods: This study took place in Sarawak, Malaysia among 11 pig farms, two slaughter houses, and three animal markets in June and July of 2017. Pig fecal, pig oral secretion, bioaerosol, and worker nasal wash samples were collected and analyzed via qRT-PCR for swine viruses. Workers were also surveyed for the nature of their occupational exposure with animals and their perceptions and use of personal protective equipment. Fisher’s Exact p-values and odds ratios were used to identify predictors of virus positivity.

Results: In all, 55 pig fecal, 49 pig oral or water, 21 bioaerosol, and 78 worker nasal wash samples were collected across 16 sites. Of these, 21 (38.2%) pig fecal, 43 (87.8%) pig oral or water, 3 (14.2%) bioaerosol, and 4 (5.1%) worker nasal wash samples were positive for PCV2 by qPCR. Porcine rotavirus C was detected in one (1.8%) pig fecal sample. No porcine rotavirus A or encephalomyocarditis virus was detected. The SYBR-based qRT-PCR assay used for PRRSv resulted in high levels of non-specific binding, omitting its inclusion in this write up. Statistically significant risk factors for PCV2 positivity among humans included having a household member with contact with pigs, farms with 1000 or more pigs, and a higher frequency of spotting rodents on the site. The personal protective equipment perceived as most effective at preventing cross-species infection and with the highest use were showering out of work and wearing dedicated boots. Among the equipment where use differed significantly from perception of efficacy were safety glasses, flu vaccination, showering out, and disposable boots.

Conclusions: Porcine circovirus has been posited as a zoonotic pathogen but limited studies have presented mixed results. Our data support the possibility of conducting a future prospective occupational study of pig workers for infection (not just nasal carriage) with this pathogen. Our data also shed light on contradictions between perception and use of personal protective equipment, stressing a need for education in farm biosecurity.