Melioidosis in Cynomolgus Macaques (Macaca fascicularis) Imported to the United States from Cambodia

Abstract

Melioidosis, a potentially fatal infectious disease of humans and animals, including nonhuman primates (NHPs), is caused by the high-consequence pathogen Burkholderia pseudomallei. This environmental bacterium is found in the soil and water of tropical regions, such as Southeast Asia, where melioidosis is endemic. The global movement of humans and animals can introduce B. pseudomallei into nonendemic regions of the United States, where environmental conditions could allow establishment of the organism. Approximately 60% of NHPs imported into the United States originate in countries considered endemic for melioidosis. To prevent the introduction of infectious agents to the United States, the Centers for Disease Control and Prevention (CDC) requires newly imported NHPs to be quarantined for at least 31 d, during which time their health is closely monitored. Most diseases of public health concern that are transmissible from imported NHPs have relatively short incubation periods that fall within the 31-d quarantine period. However, animals infected with B. pseudomallei may appear healthy for months to years before showing signs of illness, during which time they can shed the organism into the environment. Melioidosis presents diagnostic challenges because it causes nonspecific clinical signs, serologic screening can produce unreliable results, and culture isolates are often misidentified on rapid commercial testing systems. Here, we present a case of melioidosis in a cynomolgus macaque (Macaca fascicularis) that developed a subcutaneous abscess after importation from Cambodia to the United States. The bacterial isolate from the abscess was initially misidentified on a commercial test. This case emphasizes the possibility of melioidosis in NHPs imported from endemic countries and its associated diagnostic challenges. If melioidosis is suspected, diagnostic samples and culture isolates should be submitted to a laboratory in the CDC Laboratory Response Network for conclusive identification and characterization of the pathogen.

Introduction

Melioidosis is a potentially fatal disease caused by the gram-negative bacillus Burkholderia pseudomallei, an environmental bacterium found in the soil and water of tropical and subtropical regions. The exact global distribution of this bacterium is unknown; however, B. pseudomallei is currently classified as endemic in Southeast Asia, Northern Australia, Southern China, the Indian subcontinent, Africa, Puerto Rico, and parts of South America.^2,20,33 Modeling suggests that 165,000 human melioidosis cases occur globally per year, 89,000 of which are fatal, despite treatment.³³ Melioidosis is likely underreported due to nonspecific clinical signs and a lack of rapid, reliable, commercially available diagnostic tests.^22,28,51 Melioidosis occurs in humans and a wide range of animals, including nonhuman primates (NHPs) and other wild and domestic mammals, birds, and reptiles.^{3,13,16,19,26,45,47} Melioidosis is occasionally diagnosed in humans and animals in the United States, but prior to 2022, B. pseudomallei was not considered endemic because it had not been detected in the environment in the continental US.

Characteristics of melioidosis are similar in humans and animals. Transmission typically occurs when breaks in the skin are contaminated by environmental B. pseudomallei or through ingestion or inhalation. Risk of exposure increases during extreme weather events such as heavy rainfall and flooding, which allow bacteria in the soil to percolate to the surface and potentially become aerosolized.¹⁰ People who work in rice fields of endemic regions, in microbiologic laboratories, and those working or living with animals in or imported from endemic areas may be at increased risk of exposure.^{2,10,11,50,54} Direct transmission between animals and humans is rare.⁵⁰ Incubation periods are highly variable, ranging from one day to several years in humans, although in most cases, symptom onset occurs 1 to 21 d after exposure. The incubation period in animals is not definitively known, but considering the phylogenetic relatedness of primates, the incubation period in humans can reasonably be extrapolated to NHPs. Experimentally infected NHPs developed clinical signs within 1 to 2 d after respiratory exposure to B. pseudomallei.^38,52,53 In naturally infected animals, time of exposure is generally unknown, but onset of clinical signs in documented animal cases, including NHPs, ranged from 3 d to 10 y after import from endemic to nonendemic regions.^{6,12,16,18,26} Disease manifestation in humans and animals is nonspecific, with clinical signs ranging from soft tissue abscesses to pneumonia, sepsis, and death. Predisposing conditions for severe melioidosis in humans include diabetes mellitus, heavy alcohol consumption, and chronic lung disease.^10,11,50 The case fatality rate for melioidosis is 10% to 50%, but may reach 90% if sepsis develops.^34,50 Treatment requires a prolonged course of intravenous antimicrobials followed by oral administration. However, treatment may be complicated because B. pseudomallei is inherently resistant to some antimicrobials.⁵⁰ An approved vaccine is not currently available to prevent melioidosis in humans or animals.

The gold standard for diagnosis is culture of the organism from any clinical specimen. Commercially available automated identification systems may misidentify B. pseudomallei as another Burkholderia species, Pseudomonas species, Bacillus species, or other bacteria, especially in nonendemic regions where it is less likely to be considered.^28,50 In the laboratory, biosafety level (BSL)-2 conditions are recommended when inoculating potentially infectious clinical samples on culture media for diagnostic purposes. BSL-3 or animal biosafety level (ABSL)-3 conditions are recommended for handling isolates or materials suspected or known to contain B. pseudomallei.²⁷

Due to the potential for deliberate misuse with associated mass casualties and adverse effects on the economy, infrastructure, and public confidence, B. pseudomallei is classified as a Tier 1 overlap select agent by the Federal Select Agent Program (FSAP).¹⁷ The FSAP regulates the possession, use, and transfer of select agents, including B. pseudomallei. Regulation is jointly managed by Divisions at the United States Department of Agriculture (USDA) for animal cases, and the Department of Health and Human Services (HHS) for human cases. The BioThreat Team at the Texas Department of State Health Services (DSHS) Laboratory is an Advanced Reference member of the CDC Laboratory Response Network for Biologic Threats (LRN-B). The LRN network links veterinary, agriculture, public health, military, food, and water laboratories across the United States with the shared goal of rapid detection of biologic threats, whether man-made or naturally occurring. When a front-line or Sentinel laboratory initiates testing and suspects or is unable to rule out a select agent or agent of bioterrorism, they refer the sample to a reference laboratory for testing. The CDC provides standard operating procedures (SOPs) and reagents to ensure that standardized tests are performed in all LRN Reference laboratories.

Increased human and animal movement due to globalization as well as warming temperatures and severe storms associated with climate change could lead to the establishment of environmental B. pseudomallei in nonendemic regions. The presence of environmental B. pseudomallei correlates with high levels of rainfall, cloud cover, warm temperatures, and soil modified by agricultural activity.^33,35 Global warming has led to an increase in extreme rainfall events throughout the world during the 20^th and 21^st centuries, and increasing temperatures and severe weather events are expected to continue.¹⁵ Since the early 1970s, occasional instances of melioidosis have been documented in animals imported to nonendemic regions such as Europe and the United States, including NHPs, a panda, iguanas, a dog, and tropical fish.^{3,13,16,19,26,29,36,45} Infected animals, even if asymptomatic, can shed B. pseudomallei into the environment in urine, feces, and lesion exudates.⁴⁹ Therefore, importation of animals, including NHPs, infected with B. pseudomallei could introduce the organism into nonendemic areas where changing environmental conditions could allow establishment of the organism.

The Centers for Disease Control and Prevention regulates the importation of NHPs under the authority of the Public Health Service Act. These animals may be infected with zoonotic pathogens of public health concern, including B. pseudomallei. Title 42 of the Code of Federal Regulations §71.53 specifies that NHPs may only be imported by CDC-registered importers for science, education, or exhibition. CDC-registered importers must develop detailed SOPs to protect employees working with recently imported NHPs, maintain CDC-approved quarantine facilities, and undergo unscheduled site inspections and audits. Imported NHPs are held under CDC-mandated quarantine for at least 31 days, during which time they are closely monitored and tested for infectious diseases. If a disease of public health concern is identified in NHPs during quarantine, CDC, state, and local health departments respond collaboratively. The purpose of the NHP importation program is to prevent the introduction of agents of public health concern into the United States. However, the variable incubation period associated with melioidosis can mean that some NHPs appear healthy throughout the 31-d mandated quarantine period but develop clinical signs later.

The majority of NHPs imported to the United States come from regions of the world in which melioidosis is currently considered endemic.^5,33 Importers and subsequent recipients of NHPs from endemic regions should be aware of melioidosis and associated diagnostic challenges. Here, we present (to the best of our knowledge) the first published case of melioidosis in an NHP diagnosed during the CDC-mandated quarantine period.

Index Case

On December 10, 2020, a cohort of 360 purpose-bred cynomolgus macaques (Macaca fascicularis) was imported into the United States from Cambodia for use in biomedical research. The cohort was quarantined at a CDC-registered facility in Texas. All animal care and use at the facility is conducted in accordance with federal policies and guidelines and approved by the quarantine facility’s Institutional Animal Care and Use Committee (IACUC). This facility is a USDA-registered broker and has an animal care program accredited by AALAC International.

On December 30, 2020, during a routine physical examination performed on a sedated (ketamine 10 mg/kg IM) 2-y old, 1.6-kg female macaque, a 1 cm × 1 cm subdermal mass was seen on the dorsal aspect of the lumbosacral region. No other abnormalities were found, and the animal recovered from sedation uneventfully. On January 6, 2021, the mass became fluctuant and had increased in size to 2 cm × 2 cm. The animal was sedated (ketamine 10 mg/kg IM) for surgical treatment. Per the importer’s SOPs for CDC-mandated quarantine, the procedure was performed in a negative pressure anteroom of the animal holding area. Personal protective equipment (PPE) worn by the facility veterinarian and assistants included fluid-resistant Tyvek coveralls, fit tested N95 respirators, double nitrile gloves, hair bonnets, face shields, and quarantine dedicated scrubs and boots. The area around the mass was aseptically prepared, and the mass was lanced and flushed with 0.9% saline. The mass grossly appeared to be an abscess with mucopurulent contents. After the surgical procedure was completed, the table and surrounding areas were disinfected with Wexcide 128 (Wexford Labs, Kirkwood, MO). Samples of the mucopurulent material were submitted to a commercial diagnostic lab for culture and sensitivity (VRL Laboratories San Antonio, TX) and acid-fast staining (Texas Veterinary Pathologists San Antonio, TX).

On January 7, 2021, the macaque’s condition deteriorated. She became dehydrated, inappetent, and developed a left head tilt. Dexamethasone (2 mg/kg once daily × 3 d, IM), enrofloxacin (10 mg/kg once daily × 7 d, IM), penicillin procaine G (30,000 U/kg once daily × 7 d, IM), and Lactated Ringers Solution (50 mL/kg IV, once) were administered. The macaque stabilized within 2 d. On January 12, 2021, the bacterial isolate was reported as Burkholderia cepacia sensitive to trimethoprim/sulfamethoxazole (TMS). Cytology of the mucopurulent material was consistent with an abscess (pyogranulomatous inflammation) and negative for acid-fast bacteria. Treatment with TMS (5 mg/kg PO, twice daily) was initiated on January 12, 2021. The macaque responded well with resolution of the head tilt and subcutaneous abscess on January 14, 2021 and January 20, 2021, respectively. Due to the potential for misidentification of B. pseudomallei using commercially available automated systems, and because the NHP originated in a melioidosis-endemic region, the CDC requested that the bacterial isolate be submitted to the Texas DSHS Laboratory in Austin, TX for confirmation. The isolate was packaged and shipped as a diagnostic specimen in accordance with US Department of Transportation hazardous materials regulations.¹

Results/Case Outcome

Preliminary isolate identification.

VRL used 2 commercial biochemical identification systems to identify the NHP isolate, and both misidentified it as B. cepacia. The RapID NF Plus (Thermo Scientific, Waltham, MA) and the API 20NE (BioMérieux, Durham, NC) use 18 and 21 biochemical reactions, respectively, and comparison to an organism database to determine the identity of a bacterial isolate.^4,42 The API 20NE (BioMérieux, Durham, NC) gave a B. cepacia probability of 84.7%, whereas the RapID NF Plus (Thermo Scientific, Waltham, MA) gave a probability of 99.9%. Burkholderia pseudomallei is present in both databases, but misidentification has been documented.²²

Confirmatory isolate identification.

On January 20, 2021, the Texas DSHS Laboratory BioThreat Team received a pure culture isolate streaked on Sheep Blood Agar and MacConkey Agar. Aerobic culture was initiated, a Gram stain was performed, and a cell lysate containing the organism’s DNA was prepared for subsequent PCR. The LRN PCR assay that detects and can differentiate Burkholderia mallei and pseudomallei was performed. The Gram stain slide exam showed small gram-negative rods, and when later repeated with fresher growth revealed bipolar staining. The PCR result was positive for all targets, indicating that B. pseudomallei DNA was detected. These data along with phenotypic characterization of the isolate confirmed that it was B. pseudomallei.

These significant results required immediate notification to multiple partners. Upon detection of B. pseudomallei DNA by PCR, the quarantine facility, VRL, laboratory leadership, and the FBI Weapons of Mass Destruction (WMD) Coordinator were notified. The CDC Emergency Operation Center (EOC) was alerted, and electronic laboratory results were submitted to the CDC. Once the culture isolate was confirmed as B. pseudomallei, immediate notification was made to the FSAP, and SAP Form 4A Sections A and B were completed within 7 d, in accordance with FSAP policy.

After preliminary positive results were obtained and confirmatory tests were pending, a subculture of the isolate was forwarded to the CDC Bacterial Special Pathogens Branch for whole genome sequencing.

Whole genome sequencing.

DNA extraction was performed using the Promega Maxwell RSC instrument and the Maxwell RSC Cultured Cells DNA kit. Whole genome sequencing and analysis were performed at the CDC as previously described.¹⁴ Tested samples consisted of isolate BpTX2021NHP_α from the current investigation along with isolates BpTX2014NHP_α, and BpTX2015NHP_α which were recovered from the same NHP facility in Texas in 2014 and 2015 respectively. Sequences are available at NCBI under BioProject PRJNA782614.

The most common method of genetically subtyping B. pseudomallei is currently multilocus sequence typing (MLST), which is based on the comparison of segments of 7 housekeeping genes. MLST is considered to have a medium level of genetic resolution. BpTX2021NHP_α yielded a MLST result of ST 15, whereas BpTX2014NHP_α was ST 70 and BpTX2015NHP_α was ST 366, indicating that none of them were clonal to each other. A higher-resolution method based on single nucleotide polymorphisms (SNPs) of the whole genome sequence was also performed. It also indicated that the 3 isolates were not related to each other (Figure 1). SNP analysis indicated that BpTX2021NHP_α groups with other B. pseudomallei genomes with origins in Southeast Asia, which is consistent with the origin of the imported NHP.

Figure 1.

Phylogenomic single nucleotide polymorphism (SNP) tree of Burkholderia pseudomallei strains throughout the world (n = 1,695). The geographic origin of each strain, collected from the NCBI BioSample database, is colored at the tree leaves. Each of 3 strains isolated from individual Cambodian NHPs imported to the United States are shown with red branches and emphasized with black arrows to indicate phylogenetic placements in the large tree. BpTX2021NHP_Alpha was isolated from the NHP described in this report; BPTX2014NHP_Alpha and BPTX2015NHP_Alpha were imported in 2014 and 2015, respectively. All 3 NHP strains occur within East Asia; however, each is clearly distant from the others. Scale bar and substitutions are per core SNP site.

Necropsy.

Upon confirmation of B. pseudomallei, the macaque was euthanized due to concerns about zoonotic transmission, prolonged treatment time, the possibility of recurrence, and potential introduction of this Tier 1 select agent into the environment. Any imported NHP that dies or is euthanized during CDC-mandated quarantine must undergo a full postmortem examination. On January 28, 2021, a necropsy was performed on a downdraft table, and those involved wore fluid-resistant Tyvek coveralls, fit tested N95 respirators, double nitrile gloves, hair bonnets, face shields, and quarantine dedicated scrubs and boots. Gross pathology included multifocal smooth, round, cream-colored, liquid-filled lesions (6 to 10 mm in diameter) on the liver with multiple omental adhesions (Figure 2A). The spleen had a solitary, raised, off-white colored plaque (1 × 1 × 8 mm) with an omental adhesion present (Figure 2B). Multifocal, irregular, cream-colored, liquid-filled lesions (5 to 15 mm in diameter) were seen in the right middle and caudal lung lobes (Figure 2C). The spine contained a similar lesion (8 mm in diameter) between the sixth and seventh thoracic vertebra on the ventromedial aspect (Figure 2D). All other organ systems were grossly unremarkable. After the necropsy, Wexcide 128 (Wexford Labs, Kirkwood, MO) was used to disinfect the area and equipment. Formalin-fixed tissue samples were submitted to the CDC for histopathologic analysis.

Figure 2.

(A) Gross liver lesions (arrows; scale is cm). (B) Gross splenic lesion (arrow; scale is cm). (C) Gross lung lesion, right caudal lung lobe (arrow; scale is cm). (D). Gross spinal cord lesion (arrow; longitudinal section; left is cranial; scale is cm).

Histopathology, immunohistochemistry, and tissue-based molecular analysis.

Methodology included Lillie-Twort Gram and Warthin-Starry staining, immunohistochemistry using rabbit polyclonal antibody anti-Burkholderia pseudomallei at 1:1000 dilution, and a Mach 4 Universal AP Polymer Kit (Biocare Medical, Pacheco, CA) with Permanent Red Chromogen (Cell Marque/Millipore Sigma, Rocklin, CA). DNA was extracted from 16-μm paraffin-embedded lung and liver tissue sections by using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA), as described previously.²³ DNA samples were evaluated by a Burkholderia group specific PCR assay²⁴ and positive amplicons were directly sequenced by Sanger sequencing on a GenomeLab GeXP sequencer (AB SCIEX LLC, Redwood City, CA). The search for homologies to known sequences was performed by using the BLAST nucleotide database.³⁷ To ensure the quality of DNA extracts, each sample was also tested by a house-keeping gene PCR assay.⁴⁰

Significant histopathologic abnormalities (Figure 3) included multiple large abscesses with necrosis in the lungs and liver, intradermal hemorrhage and perivascular neutrophilic infiltrate at the site of the skin lesion, and bone marrow autolysis. Bacteria were not identified by Gram or Warthin-Starry staining in any of the tissue sections examined. The liver abscesses contained multifocal extracellular bacilliform, and intracellular and granular B. pseudomallei immunostaining (Figure 3D). Rare B. pseudomallei antigens were observed in the areas of lung abscesses. Liver and lung tissues were positive by the Burkholderia PCR and sequence analysis of positive amplicons showed highest identities with Burkholderia pseudomallei.

Figure 3.

Histopathologic and immunohistochemical findings in in a cynomolgus macaque (Macaca fascicularis) case of B. pseudomallei infection: (A) Low magnification of lung shows microabscess. Hematoxylin and eosin (H and E), 5× original magnification. (B) Low magnification of liver shows abscess with necrosis (*) surrounded by neutrophils, macrophage, and fibrosis. Hematoxylin and eosin (H and E), 5× original magnification. (C) High magnification of liver abscess wall shows inflammatory infiltrate composed predominantly by neutrophils and macrophages. Hematoxylin and eosin (H and E), 20× original magnification, (D) Immunostaining by using anti–Burkholderia pseudomallei antibody.

Epidemiologic Investigation.

CDC.

NHP importations are tracked in the CDC Quarantine Activity Reporting System (QARS), a secure database that records CDC public health activities at US borders. We used this database to determine that 61% of the NHPs imported to the United States from 2005 to 2020 were from regions of the world currently considered endemic for melioidosis (Table 1). At ports of entry, CDC Quarantine Public Health Officers (QPHOs) monitor the offloading of NHPs from aircraft onto transport vehicles. The QPHOs correct any regulatory or SOP noncompliance that could represent a public health risk. Noncompliance is recorded in the QARS database. Upon confirmation of melioidosis in the NHP, records of the shipment arrival and offloading were reviewed by Veterinary Medical Officers (VMOs). The records indicated that all personnel involved in offloading and transporting the shipment followed safety protocols including proper use of PPE. The QPHO who monitored the shipment also recorded that appropriate disinfection of the aircraft and other potentially contaminated surfaces had occurred. Considering the adherence to safety protocols and lack of actively draining skin lesions in the affected NHP upon importation, the potential for human exposure at the port of entry was considered minimal. Out of an abundance of caution, the CDC quarantine station staff and their supervisors were briefed on the situation and were educated about melioidosis by VMOs. The CDC also verified with the importer that the NHP caregivers had strictly adhered to biosafety protocols throughout the quarantine period at the importer’s facility. The other 359 NHPs in the cohort appeared healthy at the end of the quarantine period and were released from CDC-mandated quarantine.

Table 1.

Number of NHPs imported to the United States by country of origin from January 1, 2005, to December 31, 2020. Confirmed endemic regions are regions where B. pseudomallei has been detected in the environment. Presumed endemic regions have had human or animal cases with probable local exposure.

Country of Origin	Number NHP Imported	Percent of Total
Confirmed Endemic
Brazil	29	<0.1
Cambodia	56,644	14.5
Chinaa	147,199	37.6
Madagascar	2	<0.1
Myanmar	5	<0.1
Singapore	22	<0.1
Vietnam	23,175	5.9
Presumed Endemic
Indonesia	4,743	1.2
Panama	13	<0.1
Philippines	4,448	1.1
South Africa	2,258	0.6
All other (not currently known or presumed to be endemic)	153,055	39.1
Total	391,593	100

^a Guangxi region, Guangdong province, Hainan Province, Hong Kong

Texas Department of State Health Services Zoonosis Control.

The DSHS Zoonosis Control Branch (ZCB) central office in Austin, TX and 8 regional Zoonosis Control (ZC) offices respond to and investigate reportable zoonoses, including exotic diseases such as melioidosis. Animal infections that pose a risk to staff or the public are investigated to identify human exposures and mitigate public health risks. On January 19, 2021, the DSHS Laboratory notified ZCB of an isolate submitted by VRL Laboratories to rule out B. pseudomallei based on the recommendation of the CDC; this notification followed the usual select agent testing communication protocol between the DSHS Laboratory and ZCB. CDC notified ZCB on the same day of their suspicion of misidentification of B. pseudomallei as B. cepacia. The CDC provided primary guidance to the veterinarian treating the NHP at the quarantine facility and remained in communication with ZCB and the involved regional ZC offices during euthanasia and subsequent pathologic testing due to the public health importance of melioidosis in an imported NHP. ZCB notified the regional offices for the quarantine facility and the other laboratories involved in the VRL isolate receipt, initial positive PCR result, and subsequent culture confirmation of B. pseudomallei at the DSHS Laboratory. Once B. pseudomallei DNA was detected by the DSHS Laboratory on January 20, 2021, ZC staff verified that no laboratory exposures or breaches in PPE occurred at the quarantine facility or the laboratory facilities involved with initial testing. VRL Laboratories performed all procedures and sample manipulation under a biosafety cabinet and Texas Veterinary Pathology Associates reported no human exposures and inactivation of all potentially contaminated materials. An investigation completed by the regional ZC office for the quarantine facility confirmed that no unprotected human exposures were identified from handling the B. pseudomallei isolate or the infected NHP imported from Cambodia.

Discussion

To date, the authors are aware of 22 confirmed melioidosis cases in NHPs in the United States, including the case presented here and 5 subsequent cases identified after initial drafting of this manuscript.^{3,6,7,18,29,31,43,46} To the best of our knowledge, this is the first documented case of melioidosis diagnosed during the CDC-mandated post-importation quarantine period. The regulatory requirement for imported NHPs to remain under CDC-mandated quarantine for at least 31 d is based on the minimum time it takes to complete 3 consecutive tuberculin skin tests (TSTs) at 2-wk intervals. In addition, 31 d is longer than the incubation period for filoviruses and many other high-consequence pathogens that could be transmitted by newly imported NHPs.⁴⁴ However, the incubation period of melioidosis is highly variable, not well defined in animals, and can exceed 31 d. One report described a rhesus macaque that developed melioidosis 10 y after importation into the US.¹⁸ In an unpublished case from 2015, B. pseudomallei was isolated from a liver abscess found at necropsy in an NHP that was euthanized due to persistent lethargy and dehydration almost a year after importation and release from CDC-mandated quarantine. After the case reported here, 5 additional macaques imported in separate shipments from Cambodia developed abscesses from which B. pseudomallei was isolated several months after importation and, in 4 of the cases, after release from CDC-mandated quarantine. These cases illustrate the impracticality of holding imported NHPs in quarantine beyond the incubation period for melioidosis. In addition, serology is not a useful screening tool in animals from endemic regions due to cross-reactivity and poor correlation with active infection or development of clinical signs.^47,48 Thus, recipients of NHPs imported from regions of the world in which B. pseudomallei is endemic should be aware of the potential for melioidosis in a sick NHP, even well after the CDC-mandated quarantine period has ended.

Several factors were considered when determining the most appropriate course of action for the infected macaque. B. pseudomallei is inherently resistant to certain antimicrobials, including first-generation β-lactams, aminoglycosides, macrolides, and fluoroquinolones. In humans, treatment consists of an initial “intensive phase” of intravenous ceftazidime or a carbapenem for at least 10 to 14 d followed by an “eradication phase” of oral antibiotic therapy, usually TMS, for at least 3 to 6 mo.^34,50 Recrudescence can occur in animals and humans if the infection is not completely resolved when treatment is discontinued. Melioidosis is more likely to relapse if inappropriate antibiotics are administered or if the course of treatment is too short.^11,30,32 Rates of recrudescence or recurrence in human melioidosis patients range from 5% to 28%.^10,50

In animals, euthanasia is generally recommended instead of treatment due to concerns about zoonotic transmission, prolonged treatment time, chance of recurrence, and, in nonendemic areas, the possibility of introducing the agent into the environment.^47,49 In 2013, an imported macaque developed a draining tract in the skin that resolved after a 3-mo course of antimicrobials, but 2 mo later it succumbed to disseminated melioidosis.²⁹ In our case, the macaque had clinically improved and the skin lesion had resolved on antibiotics, but considering the factors just discussed, the decision was made to euthanize. Postmortem diagnostics confirmed B. pseudomallei-associated abscesses in at least the lungs and liver, despite the macaque having received antimicrobial treatment for 3 wk and the resolution of its initial skin lesion prior to euthanasia.

In the current case, the decision to release the remainder of the cohort from CDC-mandated quarantine was made after consideration of several factors. The other macaques had not shown any clinical signs of disease during the quarantine period and were apparently healthy. The animals were singly housed in quarantine, so none of the other NHPs had direct contact with the sick macaque. The infection is rarely spread from animal to animal; B. pseudomallei is most commonly transmitted via contamination of a skin wound from the environment.⁵⁰ The environment within the facility was not conducive to pathogen persistence due to quarantine protocols for disinfection and treatment of waste as biohazardous material. The incubation period for melioidosis is so variable that determination of an appropriate timeline for extended quarantine was not feasible. Serologic testing of the cohort was considered, but insufficient evidence indicates that a negative or positive titer predicts active infection or development of melioidosis. The CDC advised the NHP importer to notify recipients of the cohort that the NHPs may have been exposed to B. pseudomallei in Cambodia and to be aware of the variable incubation period, range of clinical signs, and diagnostic challenges associated with melioidosis. The importer was provided educational materials created by the CDC to distribute to their clients. The CDC distributed a letter of information to state public health officials, NHP importers, and veterinarians who work in the NHP industry to raise awareness of the potential for melioidosis in imported NHPs. Although melioidosis is not reportable to the World Organization for Animal Health, the CDC notified Cambodian animal health officials of the case reported here and of the subsequent cases in NHPs imported from Cambodia. The CDC has not received any report of clinical illness in the cohorts to date, although once the NHPs are released from quarantine, reporting animal illness to the CDC is not required. Registered importers must report signs of zoonotic illness in quarantine staff to the CDC; to date, human illness has not been reported in connection with the NHP cases.

This case highlights the importance of confirmatory testing through a CDC LRN member laboratory if clinical signs of melioidosis occur in animals imported from endemic regions. In the reported case, the isolate was initially identified as B. cepacia, which is endemic in the United States and less pathogenic than B. pseudomallei. The gold standard for diagnosing melioidosis is culture and isolation of the causative agent. Rapid, automated, commercially available identification systems are known to occasionally misidentify B. pseudomallei as another species or even genus of bacteria (for example, Pseudomonas spp or Bacillus spp).^22,28 In 2021, after the case reported here, a second cynomolgus macaque imported from Cambodia was reported with peritoneal abscesses from which a commercial laboratory isolated E. coli and Enterococcus spp.; a reference laboratory subsequently identified B. pseudomallei, (personal communication, CDC Bacterial Special Pathogens Branch). Awareness of possible misidentifications and subsequent referral of the isolate to rule out B. pseudomallei are important to ensure that cases are detected, especially in nonendemic areas in which melioidosis may not be initially considered.⁵¹ In addition, commercially available PCR for tissue samples may have low sensitivity compared with PCR of pure cultures and may produce false negative results.²⁸ LRN member laboratories such as the Texas DSHS Laboratory in Austin are equipped to safely and accurately identify emerging and high-consequence pathogens. Anyone working with NHPs or other animals imported from B. pseudomallei endemic regions must maintain awareness of melioidosis and the associated diagnostic challenges.

The introduction and environmental persistence of B. pseudomallei in nonendemic regions could potentially develop through international travel and animal movement. The Jardin des Plantes, a botanical garden and zoo in Paris, France, was the site of a melioidosis outbreak that resulted in animal and human deaths in the 1970s. B. pseudomallei was thought to have been introduced to the zoo environment by a panda imported from China. The pathogen spread through the environment in and around Paris and could be detected in the soil for at least 6 y.^12,16,36 Environmental conditions in the southern United States, including coastal regions of Texas, are similar to those in regions of the world in which B. pseudomallei is endemic.^33,41 A soil survey conducted in 2012 identified diverse Burkholderia spp. in all 3 states from which soil was sampled (Arizona, Florida, and Louisiana); B. pseudomallei was not detected, but the environment in these areas does support survival of other species in the same genus.²⁵ In 2022, B. pseudomallei was identified in soil and water in southern Mississippi, representing the first time the bacterium has been isolated from the environment in the continental United States.⁸ In Texas, human cases of melioidosis have occurred after decades had passed since international travel; the isolates were genetically similar to isolates from other cases with confirmed origin in the Americas.^9,21,39 This raises the question as to whether B. pseudomallei could also be present in US soil outside of Mississippi, but until it is isolated from the environment, most of the United States is considered nonendemic, and vigilance is critical to preventing its introduction via imported animals.

Those who import or receive imported animals from regions of the world with endemic melioidosis or from nonendemic countries that import NHPs from endemic regions should be aware of the disease and consider it a differential in animals that display compatible clinical signs, regardless of the time that has passed since importation. For staff safety, if B. pseudomallei is suspected, the submitter should notify the laboratory so that appropriate safety precautions can be taken, such as performing all manipulations in a biosafety cabinet. Diagnostic laboratories should be aware of the challenges presented by B. pseudomallei identification and should consider confirmatory identification by an LRN member laboratory if B. cepacia or other Burkholderia spp, Pseudomonas spp, or colonies that appear as contaminants are isolated from an imported animal. Although melioidosis is not a nationally notifiable disease, laboratory confirmation of B. pseudomallei isolates requires reporting to the Federal Select Agent Program for human samples or Animal and Plant Health Inspection Service (APHIS) for animal samples. Reporting confirmed animal and human cases to state public health officials is imperative to understanding the epidemiology in the United States so that appropriate investigations can be conducted. The addition of melioidosis to nationally notifiable human and animal disease lists should be considered.

Disclaimer

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the CDC.