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A rapid review was performed to determine (1) the number and causes of reported laboratory-acquired infections (LAI) in the Asia-Pacific region; (2) their significance and threat to the community; (3) the primary risk factors associated with LAIs; (4) the consequences in the event of a LAI or pathogen escape; and (5) to make general recommendations regarding biosafety practices for diagnosis and research in the Asia-Pacific region. A search for LAI and zoonoses in the Asia-Pacific region using online search engines revealed a relatively low number of reports. Only 27 LAI reports were published between 1982 and 2016. The most common pathogens associated with LAIs were dengue virus, Arthroderma spp., Brucella spp., Mycobacterium spp., Rickettsia spp., and Shigella spp. Seventy-eight percent (21 out of 27 LAI reports) occurred in high-income countries (i.e., Australia, Japan, South Korea, Singapore, and Taiwan) where laboratories were likely to comply with international biosafety standards. Two upper-middle income countries (China (2), and Malaysia (2)) and one lower-middle income country (India (2)) reported LAI incidents. The majority of the reports (fifty-two percent (14/27)) of LAIs occurred in research laboratories. Five LAI reports were from clinical or diagnostic laboratories that are considered at the frontier for zoonotic disease detection. Governments and laboratories in the Asia-Pacific region should be encouraged to report LAI cases as it provides a useful tool to monitor unintended release of zoonotic pathogens and to further improve laboratory biosafety. Non-reporting of LAI events could pose a risk of disease transmission from infected laboratory staff to communities and the environment. The international community has an important and continuing role to play in supporting laboratories in the Asia-Pacific region to ensure that they maintain the safe working environment for the staff and their families, and the wider community.

INTRODUCTION: Global concerns over emerging and transboundary infectious zoonotic diseases have increased disease diagnostic demands, especially in the veterinary sector. In developing or newly developed countries where the sector often works under limited capacity, biosafety and biosecurity are unlikely to be high-priority issues. A recent development program supported by the Biological Threat Reduction Program of the Defense Threat Reduction Agency funded by the US government aimed to increase biosafety and biosecurity measures of government veterinary diagnostic and research laboratories in Thailand. OBJECTIVE: The purpose of this article is to identify biosafety and biosecurity challenges, opportunities, and recommendations. METHODS: Eleven government laboratory centers were assessed against the Biosafety in Microbiological and Biomedical Laboratories (BMBL) biosafety level 2 (BSL-2) requirements checklist. The BMBL assessment outcomes were then combined with the outcomes of discussion sessions, and the results of pre- and post-test questionnaires conducted during biosafety assessment workshops and self-evaluation reports using the Food and Agriculture Organization Biosafety Laboratory Mapping Tool of each laboratory center were reviewed and summarized. RESULTS: Despite established national policies on laboratory biosafety and biosecurity, major challenges included (1) harmonization and enforcement of these policies, especially at the regional level, and (2) engagement of personnel in implementations of biosafety and biosecurity measures. CONCLUSION: Consistent biosafety policy and allocated resources together with regular training are required to develop sustainable biosafety and biosecurity at the national level. Collaboration between regional countries, international organizations, and donors is essential for improving biosafety and biosecurity on a global scale through setting regional priorities, enacting regulatory standards, and providing technical and financial support.

We performed indirect immunofluorescence assays (IFAs) to compare levels of IgM and IgG antibodies to Orientia tsutsugamushi and Rickettsia typhi in admission-phase serum samples and filter paper blood spots (assayed immediately and stored at 5.4°C and 29°C for 30 days) collected on the same day from 53 adults with suspected scrub typhus and murine typhus admitted to Mahosot Hospital Vientiane, Lao People's Democratic Republic. The sensitivities and specificities of admission-phase filter paper blood spots in comparison to paired sera were between 91% and 95% and 87% and 100%, respectively, for the diagnosis of scrub typhus and murine typhus. The classification of patients as having or not having typhus did not significantly differ after storage of the blood spots for 30 days (P > 0.4) at 5.4°C and 29°C. Because filter paper blood samples do not require sophisticated and expensive storage and transport, they may be an appropriate specimen collection technique for the diagnosis of rickettsial disease in the rural tropics.

Pig rearing is an important income source in the Lao People's Democratic Republic (PDR), with many smallholder farmers using traditional free-range pig production systems. Despite the potentially significant health risks posed by pig production regarding pig-associated zoonoses, information on the sociocultural drivers of these zoonoses is significantly lacking. This review summarises the existing sociocultural knowledge on eight pig-associated zoonoses suspected to be endemic in Southeast Asia: brucellosis, Q fever (Coxiella burnetii), trichinellosis, hepatitis E virus, leptospirosis, Japanese encephalitis, Streptococcus suis and Taenia solium taeniasis-cysticercosis. It summarises current knowledge on these diseases grouped according to their clinical manifestations in humans to highlight the propensity for underreporting. A literature search was conducted across multiple databases for publications from 1990 to the present day related to the eight pig-associated zoonoses and the risk and impact connected with them, with Lao PDR as a case study. Many of these pig-associated zoonoses have similar presentations and are often diagnosed as clinical syndromes. Misdiagnosis and underreporting are, therefore, substantial and emphasise the need for more robust diagnostics and appropriate surveillance systems. While some reports exist in other countries in the region, information is significantly lacking in Lao PDR with existing information coming mainly from the capital, Vientiane. The disease burden imposed by these zoonoses is not only characterised by morbidity and mortality, but directly impacts on livelihoods through income reduction and production losses, and indirectly through treatment costs and lost work opportunities. Other factors crucial to understanding and controlling these diseases are the influence of ethnicity and culture on food-consumption practices, pig rearing and slaughter practices, hygiene and sanitation, health-seeking behaviours and, therefore, risk factors for disease transmission. Published information on the knowledge, attitudes and beliefs of people regarding pig zoonoses and their risk factors is also extremely limited in Lao PDR and the broader Southeast Asian region. The need for more transdisciplinary research, using a One Health approach, in order to understand the underlining social determinants of health and their impacts on health-seeking behaviours, disease transmission and, ultimately, disease reporting, cannot be more emphasized. ; Peer Review

The increasing intensification of pork production in southeast Asia necessitates an urgent requirement to better understand the dual impact of pig-associated zoonotic disease on both pig production and human health in the region. Sharing porous borders with five countries and representing many regional ethnicities and agricultural practices, Lao People's Democratic Republic (Lao PDR) appears well placed to gauge the levels of pig-associated zoonoses circulating in the wider region. Despite this, little is known about the true impact of zoonotic pathogens such as leptospirosis, Trichinella, hepatitis E virus (HEV), Japanese encephalitis (JE), and Taenia solium on human health and livestock production in the country. A comprehensive review of the published prevalences of these five pig-associated zoonoses in Lao PDR has demonstrated that although suspicion remains high of their existence in pig reservoirs across the country, epidemiological data are scarce; only 31 epidemiological studies have been undertaken on these diseases in the past 25 years. A greater understanding of the zoonoses prevalence and subsequent risks associated with pork production in the southeast Asian region could help focus public health and food safety interventions at key points along the value chain, benefiting both livestock producers and the broader animal and human health systems in the region.

A national animal disease surveillance network initiated by the Lao PDR government is adopted and reinforced by a joint research project between the National Animal Health Laboratory (NAHL), the Department of Livestock and Fisheries (DLF), and the Mahidol Oxford Tropical Medicine Research Unit (MORU). The network is strengthened by staff training and practical exercises and is utilised to provide zoonotic or high-impact disease information on a national scale. Between January and December 2020, large ruminant samples are collected monthly from 18 abattoirs, one in each province, by provincial and district agriculture and forestry officers. The surveillance network collected a total of 4247 serum samples (1316 buffaloes and 2931 cattle) over this period. Samples are tested for antibodies against Brucella spp., Coxiella burnetii (Q fever) and Foot-and-Mouth Disease Non-Structural Protein (FMD NSP) using commercial ELISA kits and the Rose Bengal test. Seroprevalences of Q fever and brucellosis in large ruminants are low at 1.7% (95% CI: 1.3, 2.1) and 0.7% (95% CI: 0.5, 1.0) respectively, while for FMD NSP it is 50.5% (95% CI: 49.0, 52.0). Univariate analyses show differences in seroprevalences of Q fever between destination (abattoir) province (p-value = 0.005), province of origin (p-value = 0.005), animal type (buffalo or cattle) (p-value = 0.0008), and collection month (p-value = 3.4 × 10(−6)). Similar to Q fever, seroprevalences of brucellosis were significantly different for destination province (p-value < 0.00001), province of origin (p-value < 0.00001), animal type (p-value = 9.9 × 10(−5)) and collection month (p-value < 0.00001), plus body condition score (p-value = 0.003), and age (p-value = 0.007). Additionally, risk factors of the FMD NSP dataset include the destination province (p-value < 0.00001), province of origin (p-value < 0.00001), sex (p-value = 7.97 × 10(−8)), age (p-value = 0.009), collection date (p-value < 0.00001), and collection month (p-value < 0.00001). Spatial ...

BACKGROUND: Rabies is a fatal viral disease that continues to threaten both human and animal health in endemic countries. The Lao People's Democratic Republic (Lao PDR) is a rabies-endemic country in which dogs are the main reservoir and continue to present health risks for both human and animals throughout the country. METHODS: Passive, laboratory-based rabies surveillance was performed for suspected cases of dog rabies in Vientiane Capital during 2010-2016 and eight additional provinces between 2015-2016 using the Direct Fluorescent Antibody Test (DFAT). RESULTS: There were 284 rabies positive cases from 415 dog samples submitted for diagnosis. 257 cases were from Vientiane Capital (2010-2016) and the remaining 27 cases were submitted during 2015-2016 from Champassak (16 cases), Vientiane Province (4 cases), Xieng Kuang (3 cases), Luang Prabang (2 cases), Saravan (1 case), Saisomboun (1 case) and Bokeo (1 case). There was a significant increase in rabies cases during the dry season (p = 0.004) (November to April; i.e., <100mm of rainfall per month). No significant differences were noted between age, sex, locality of rabies cases. CONCLUSION: The use of laboratory-based rabies surveillance is a useful method of monitoring rabies in Lao PDR and should be expanded to other provincial centers, particularly where there are active rabies control programs.

SIMPLE SUMMARY: An abattoir based surveillance system was implemented in the Lao People's Democratic Republic to determine the prevalence of two diseases that can spread between animals and humans: Brucellosis and Q fever. A total of 683 cattle and buffalo samples were collected from abattoirs in six selected provinces between March–December 2019. Laboratory diagnostic tests to detect antibodies against both diseases were performed and the number of animals that tested positive for either disease was relatively low. However, extensive animal movement within the country was also identified, which has the potential to increase the risk of spreading disease within and between countries. Monitoring of high impact animal/human diseases assists pathogen surveillance and the country's food security. This study highlights the importance of ongoing animal health surveillance and the need to find cost-effective approaches for its long-term sustainability. ABSTRACT: Although animal health surveillance programmes are useful for gaining information to help improve global health and food security, these programmes can be challenging to establish in developing economies with a low-resource base. This study focused on establishing a national surveillance system initiated by the Lao PDR government using a passive surveillance system of abattoir samples as a pilot model, and to gain information on contagious zoonoses, particularly Q fever and brucellosis, in the large ruminant population. A total of 683 cattle and buffalo samples were collected from six selected provinces of Lao PDR between March–December 2019. Out of 271 samples tested, six samples (2.2%, 95% confidence interval (CI) of 1.0, 4.8) were positive in the Q fever antibody ELISA test. Only one sample (out of 683; 0.2%, 95% CI 0.0, 0.8) tested positive to the Brucella antibody ELISA test. Seroprevalence of these important zoonoses in Lao PDR were relatively low in cattle and buffaloes; however, extensive animal movement within the country was identified which could ...

African Swine Fever (ASF) is a transboundary animal disease of pigs and wild suids that appeared in Lao People's Democratic Republic (Lao PDR) in mid-2019, having spread across China and Vietnam in the months prior. Despite the scale of the Asian ASF pandemic and the availability of pen-side rapid diagnostic tests (RDT) on the market, few locally produced and easily available ASF RDTs have been evaluated for diagnostic accuracy. In this study, an ASF antigen detection RDT from Shenzhen Lvshiyuan Biotechnology Co. Ltd was evaluated using clinical field samples submitted to the National Animal Health Laboratory (NAHL) from ASF suspect cases between June and December 2019 in Lao PDR. Positive (n = 57) and negative (n = 50) samples of whole blood, serum and haemolysed serum were assessed by RDT and PCR, with the latter used as the gold standard reference comparator. Overall the RDT had a diagnostic sensitivity (DSe) of 65%, 95% CI [51 - 77] and diagnostic specificity (DSp) of 76%, 95% CI [62 - 87]. The RDT demonstrated improved performance on samples with lower PCR cycle threshold (ct) values with each additional cycle reducing the odds of the RDT returning a positive by 17% relative to the previous cycle, 95% CI [8% - 28%] (P < 0.01). While this test shows promise for field application, complete validation of diagnostic accuracy requires a larger sample size.

African Swine Fever (ASF) is a transboundary animal disease of pigs and wild suids that appeared in Lao People's Democratic Republic (Lao PDR) in mid-2019, having spread across China and Vietnam in the months prior. Despite the scale of the Asian ASF pandemic and the availability of pen-side rapid diagnostic tests (RDT) on the market, few locally produced and easily available ASF RDTs have been evaluated for diagnostic accuracy. In this study, an ASF antigen detection RDT from Shenzhen Lvshiyuan Biotechnology Co. Ltd was evaluated using clinical field samples submitted to the National Animal Health Laboratory (NAHL) from ASF suspect cases between June and December 2019 in Lao PDR. Positive (n = 57) and negative (n = 50) samples of whole blood, serum and haemolysed serum were assessed by RDT and PCR, with the latter used as the gold standard reference comparator. Overall the RDT had a diagnostic sensitivity (DSe) of 65 %, 95 % CI [51–77] and diagnostic specificity (DSp) of 76 %, 95 % CI [62–87]. The RDT demonstrated improved performance on samples with lower PCR cycle threshold (ct) values with each additional cycle reducing the odds of the RDT returning a positive by 17 % relative to the previous cycle, 95 % CI [8 %–28 %] (P < 0.01). While this test shows promise for field application, complete validation of diagnostic accuracy requires a larger sample size.

In the Lao People's Democratic Republic (Laos), rickettsial infections, including scrub and murine typhus, account for a significant burden of fevers. The Mahosot Hospital Microbiology Laboratory in Vientiane, Laos, routinely performs rickettsial isolation from hospitalized patients with suspected rickettsioses using mammalian cell culture systems. We review the clinical and laboratory factors associated with successful Orientia tsutsugamushi and Rickettsia typhi isolations from this laboratory over a period of 6 years between 2008 and 2014. The overall isolation success was 7.9% for all samples submitted and 17.3% for samples for which the patient had a positive O. tsutsugamushi or R. typhi rapid diagnostic test (RDT), serology, or PCR. The frequency of successful isolation was highest for samples submitted in November, at the end of the wet season (28.3%). A longer median duration of reported illness, a positive result for a concurrent Orientia or Rickettsia spp. quantitative PCR, and the use of antibiotics by the patient in the week before admission were significantly associated with isolation success (P < 0.05). Buffy coat inoculation and a shorter interval between sample collection and inoculation in the laboratory were associated with a higher frequency of isolation (both P < 0.05). This frequency was highest if cell culture inoculation occurred on the same day as blood sample collection. Factors related to the initial rickettsial bacterial concentration are likely the main contributors to isolation success. However, modifiable factors do contribute to the rickettsial isolation success, especially delays in inoculating patient samples into culture.

African swine fever virus (ASFV) causes a deadly disease of pigs which spread through southeast Asia in 2019. We investigated one of the first outbreaks of ASFV in Lao People's Democratic Republic amongst smallholder villages of Thapangtong District, Savannakhet Province. In this study, two ASFV affected villages were compared to two unaffected villages. Evidence of ASFV-like clinical signs appeared in pig herds as early as May 2019, with median epidemic days on 1 and 18 June in the two villages, respectively. Using participatory epidemiology mapping techniques, we found statistically significant spatial clustering in both outbreaks (p < 0.001). Villagers reported known risk factors for ASFV transmission – such as free-ranging management systems and wild boar access – in all four villages. The villagers reported increased pig trader activity from Vietnam before the outbreaks; however, the survey did not determine a single outbreak source. The outbreak caused substantial household financial losses with an average of nine pigs lost to the disease, and Monte Carlo analysis estimated this to be USD 215 per household. ASFV poses a significant threat to food and financial security in smallholder communities such as Thapangtong, where 40.6% of the district's population are affected by poverty. This study shows ASFV management in the region will require increased local government resources, knowledge of informal trader activity and wild boar monitoring alongside education and support to address intra-village risk factors such as free-ranging, incorrect waste disposal and swill feeding.

Pigs in Lao People's Democratic Republic are important for income and food security, particularly in rural households. The majority of pigs are reared in smallholder systems, which may challenge the implementation of any disease control strategies. To investigate risk factors for pig production diseases in such farming systems in the country a serological survey was conducted during 2011. A total of 647 pigs were sampled, accounting for 294 households in Luang Prabang and 353 in Savannakhet province representing upland and lowland, respectively. The results demonstrated that pigs in Lao PDR had antibodies against erysipelas (45.2%), CSF (11.2%), PRRS (8.6%), FMD O (17.2%) and FMD Asia 1, (3.5%). Differences in the housing systems influenced disease risk, for example, penned pigs had reduced odds of FMD and CSF, compared to those in scavenger systems. Pigs owned by farms using a sanaam (a communal area where pigs are kept for some time of the year) had 3.93 (95% confidence interval (CI): 1.09–14.7) times the odds of having pigs seropositive for FMD. Farms on which sudden piglet deaths had been experienced were more likely to have pigs seropositive for FMD O and erysipelas. These diseases constrain the development of village farming and the wider livestock industry due to their impact on productivity and trade. Vaccination coverage for FMD and CSF was low and there was a lack of national funding for livestock disease control at the time of the study. Further investigation into sustainable low-cost control strategies for these pathogens is warranted.

In Lao People's Democratic Republic pigs are kept in close contact with families. Human risk of infection with pig zoonoses arises from direct contact and consumption of unsafe pig products. This cross-sectional study was conducted in Luang Prabang (north) and Savannakhet (central-south) Provinces. A total of 59 villages, 895 humans and 647 pigs were sampled and serologically tested for zoonotic pathogens including: hepatitis E virus (HEV), Japanese encephalitis virus (JEV) and Trichinella spiralis; In addition, human sera were tested for Taenia spp. and cysticercosis. Seroprevalence of zoonotic pathogens in humans was high for HEV (Luang Prabang: 48.6%, Savannakhet: 77.7%) and T. spiralis (Luang Prabang: 59.0%, Savannakhet: 40.5%), and lower for JEV (around 5%), Taenia spp. (around 3%) and cysticercosis (Luang Prabang: 6.1, Savannakhet 1.5%). Multiple correspondence analysis and hierarchical clustering of principal components was performed on descriptive data of human hygiene practices, contact with pigs and consumption of pork products. Three clusters were identified: Cluster 1 had low pig contact and good hygiene practices, but had higher risk of T. spiralis. Most people in cluster 2 were involved in pig slaughter (83.7%), handled raw meat or offal (99.4%) and consumed raw pigs' blood (76.4%). Compared to cluster 1, cluster 2 had increased odds of testing seropositive for HEV and JEV. Cluster 3 had the lowest sanitation access and had the highest risk of HEV, cysticercosis and Taenia spp. Farmers which kept their pigs tethered (as opposed to penned) and disposed of manure in water sources had 0.85 (95% CI: 0.18 to 0.91) and 2.39 (95% CI: 1.07 to 5.34) times the odds of having pigs test seropositive for HEV, respectively. The results have been used to identify entry-points for intervention and management strategies to reduce disease exposure in humans and pigs, informing control activities in a cysticercosis hyper-endemic village.