Clostridium difficile infection in the Lao People’s Democratic Republic: first isolation and review of the literature

Background Current knowledge of the epidemiology of Clostridium difficile infection in Asia, and in particular the Greater Mekong Subregion, is very limited. Only a few studies from Thailand and Vietnam have been reported from the region with variable testing methods and results, and no studies from Lao People’s Democratic Republic (PDR). Therefore we investigated the presence of C. difficile in a single centre in the Lao PDR and determined the ribotypes present. Method Seventy unformed stool samples from hospital inpatients at Mahosot Hospital, Vientiane, were tested for the presence of C. difficile using selective differential agar and confirmed by latex agglutination. C. difficile isolates were further characterised by ribotyping and toxin gene detection. Results C. difficile was isolated from five of the 70 patients, and five different ribotypes were identified (014, 017, 020, QX 107 and QX 574). Conclusion This is the first isolation of C. difficile from human stool samples in the Lao PDR. These results will add to the limited amount of data on C. difficile in the region. In addition, we hope this information will alert clinicians to the presence of C. difficile in the country and will help inform future investigations into the epidemiology and diagnosis of C. difficile in Lao PDR.


Background
Clostridium difficile is an anaerobic, Gram-positive, spore forming bacterium that causes antibiotic-associated diarrhoea which can vary from mild and self-limiting to serious manifestations including pseudomembranous colitis [1]. C. difficile infection (CDI) is predominantly healthcare-associated although increasing numbers of community-acquired infections have been reported [2,3]. Some strains of C. difficile have the ability to produce three toxins -toxin A, toxin B and a binary cytolethal distending toxin (CDT), and only strains that can produce at least one of these toxins cause disease [4].
C. difficile is widely recognized as an important diarrheal pathogen in North America and Europe. Epidemics have occurred in both these regions, resulting in the development of guidelines on active surveillance of infection, laboratory diagnosis, management and infection prevention and control, and a need for better antibiotic stewardship within health care facilities, the community and animal production facilities [5]. In contrast, limited information on CDI is available in Asia [6]. Reports from the region are highly variable with respect to the prevalence, laboratory diagnostic methods and circulating ribotypes (RT). Not all countries in the region have reported CDI, and comparisons within the region are difficult to make [6].
Despite a number of reports of CDI in the neighbouring countries of Thailand [7] and China [8], there have been no published reports on the isolation of C. difficile in Lao People's Democratic Republic (Lao PDR, Laos). Cephalosporins, particularly ceftriaxone, are used extensively in hospitals in Laos, which may carry an associated risk of CDI. We therefore investigated the prevalence of C. difficile from stool samples from hospitalised patients from a single centre within Laos and characterised all C. difficile isolates.

Methods
The investigation was conducted in the Microbiology Laboratory at Mahosot Hospital during September and October 2013. Mahosot Hospital is a 350-bed government hospital in the Lao capital, Vientiane, which houses a range of medical and surgical units. There were 21,549 people admitted to the hospital during 2013 and 3540 people admitted during the months of September and October.
Routine testing of faeces in the Mahosot Hospital Microbiology Laboratory consists of microscopy for white cells, and culture for Salmonella and Shigella species only. Vibrio species are sought using selective media (thiosulfatecitrate-bile salts-sucrose agar; TCBS agar) when indicated by clinical or epidemiological features. The laboratory does not routinely undertake any anaerobic culture or attempt to detect Campylobacter species or viral enteric pathogens, whilst microscopy for parasites is conducted in a separate laboratory. Relatively small numbers of stool samples are sent to the laboratory. However, because of concern that C. difficile may be unrecognised in Laos, methods for C. difficile culture and identification were established as part of routine stool examination for a 2 month period. Patients gave oral consent for the collection of stool samples for diagnosis of their illness.
A memo (in Lao language) was sent to the medical teams in Mahosot Hospital notifying them of the introduction of C. difficile diagnostic techniques. The following details were obtained for each specimen: date of specimen collection, patient age and gender, clinical diagnosis as listed on the test request form, the presence of stool white cells on microscopy and the results of Salmonella/Shigella culture. Other microbiologic details were noted if present; e.g. the presence of faecal parasites on microscopy. Unfortunately it was not possible to obtain a reliable history for prior antibiotic use for every patient.
Unformed faecal specimens were directly plated onto half plates of selective differential medium (ChromID C. difficile agar, bioMerieux, France) and streaked for single colonies. Plates were incubated anaerobically (BD GasPak™ EZ Anaerobic Container System, Becton Dickinson, USA) for 48 h before examination for colonies typical of C. difficile (black, spreading, medusa-head colonies with a characteristic odour). Suspicious colonies were tested by latex agglutination according to the manufacturer's instructions (Oxoid C. difficile latex test kit, Oxoid, UK) to confirm their identity. A control strain of C. difficile (C. difficile ATCC 9689) was used to validate test results. Isolates were stored at −80°C until shipment to Perth, Australia, for confirmation of identity, testing for the presence of toxin genes (tcdA, tcdB, and cdt) and ribotyping as described previously targeting the 16S-23S rRNA gene using primers 5′-CGTGGGGTGAAGTCGTAACAAGG-3′ (positions 1445 to 1466 of the 16S rRNA gene) and 5′-GCGCCCTT TGTAGCTTGACC-3′ (positions 20 to 1 of the 23S rRNA gene) [9]. Antimicrobial susceptibility testing was also performed for moxifloxacin, metronidazole, clindamycin and vancomycin using E-tests (Biomérieux, Marcy-l'Etoile, France) with minimum inhibitory concentration (MIC) results interpreted according to current US Clinical and Laboratory Standards Institute (CLSI) guidelines.

Results
A total of 86 faecal samples were received during the 2 month period, 70 of which were available for C. difficile culture. The median age was 35 years with a range of 240 days to 89 years. There were 31 females and 39 males. Of the 70 patients included in the study, 26 were reported to have diarrhoea, 14 had suspected typhoid fever, five had fever of unknown etiology and two had suspected melioidosis. Salmonella spp. were isolated from six of the 70 samples and five were positive for C. difficile by culture and latex agglutination. Three of the five isolates of C. difficile were positive for the tcdB gene with two also tcdA positive by polymerase chain reaction (PCR). There were five different RTs identified-RTs 014, 017, 020, QX 107 and QX 574, with the latter two isolates being non-toxigenic (Table 1). All isolates were susceptible to moxifloxacin, metronidazole and vancomycin. Four of the five isolates were clindamycin intermediate and isolate the RT QX107 isolate was clindamycin resistant with an MIC of 8 μg/ml.

Discussion
This is the first report, as far as we have been able to establish, of the isolation of C. difficile from diarrheal stool specimens in Laos. In a study of the etiology of diarrhoea in Vientiane from 1996 to 1997, the presence of C. difficile was not investigated [10], and there have been no clinical reports of CDI in Laos, of which awareness amongst doctors appears low. However, increasing awareness of this pathogen is leading to increased testing and improved surveillance elsewhere in Asia [6]. It is not possible to be certain that C. difficile was a primary pathogen in all five patients given the presence of Salmonella sp. in two patients and the absence of toxin genes in two of the C. difficile isolates. Furthermore, two of the isolates were from 1-year-old children. C. difficile is known to cause asymptomatic colonization in infants under 2 years of age, so the C. difficile in these two cases may not have been responsible for causing symptoms [11].
Although many studies have been published on C. difficile epidemiology worldwide, few are from Asia, and in particular the Greater Mekong Subregion. Reports from Thailand (Bangkok) predominate, with only a single report from Vietnam ( Table 2). To date there are no published data on C. difficile infection and epidemiology from Laos, Cambodia or Myanmar showing the need for further research in these countries. The incidence of C. difficile infection in the different groups of patients listed in Table 2 ranged from 6.5% to 44%. Different diagnostic techniques were used for these studies, making comparisons difficult. Toxin enzyme immunoassay (EIA) tests alone, as used in two of these studies [12,13], have a lower sensitivity compared to EIA paired with PCR [14,15]. Three of the earliest Thailand studies also only looked at toxin A or the tcdA gene [12,13,16]. Due to the high regional prevalence of RT 017, which does not produce toxin A, it is possible that the true incidence of CDI has been underestimated in these studies [6].
All of the ribotypes detected in this current study have been reported elsewhere. RTs 014 and 020 have been isolated in several studies from Thailand and China and were both isolated in this study [8,17]. Due to the high similarity of these two RTs, they are often reported in studies as the 'RT 014/020 group'. One RT 017 isolate was identified in our study. This is a prevalent RT in many countries in Asia and the predominant RT found in Thailand, China and South Korea [8,17,18]. Two isolates of RT QX 107 were recently described for the first time from Thailand [19] with our isolate being the third detected in the region. This could suggest that this RT is of Asian origin. The RT QX 574 isolated from this study may also be unique to the region, as the only other country the strain has been isolated from is Indonesia (T. Riley, unpublished data). There were no hypervirulent RT 027 or RT 078 isolated from our small number of patients. While both RT 027 and RT 078 have been widely reported from Europe and North America [20][21][22], there have only been sporadic reports from Asia, including recent reports from mainland China [18,23,24]. The spread of these RTs into mainland China could lead to further dissemination within South East Asia. RT 078 is more commonly associated with community-acquired C. difficile infection; hence hospital-based studies, as most of the studies in this region have been, might miss cases caused by this RT [25].
In Europe and the USA, the majority of C. difficile cases are thought to result from person-to-person spread. Antimicrobial resistant bacteria have become an established problem globally, and in particular in Asia, as a result of widespread overuse and misuse of antibiotics [26,27]. This is also important in the promotion of antibioticassociated diarrhoea and CDI. The use of clindamycin, third generation cephalosporins, penicillins and fluoroquinolones greatly increases the risk of CDI [28], with the third generation cephalosporins causing the highest attributable risk due to their frequent use in hospitals [29]. One study showed that two thirds of patients with CDI had received a cephalosporin during the two month period before diagnosis [30]. The use of cephalosporins, particularly ceftriaxone, is increasing in Laos, with a 50% increase in ceftriaxone usage seen in Mahosot Hospital since 2011 (Mahosot Hospital, unpublished data). Furthermore, nearly 50% of patients admitted to Mahosot Hospital have evidence of having received antibiotics prior to admission [27]. This highlights the need for enhanced antimicrobial stewardship if further increases in CDI are to be avoided.
Given the prevalence of factors which promote CDI, and the evidence that CDI occurs at similar rates in Asia as in other continents [6], we believe that CDI is likely to be an under-recognised cause of diarrhoea in Laos and adjoining countries. There are several reasons for this under-diagnosis, including low clinician awareness of CDI [31], limited access to diagnostic services, a disincentive to investigate diarrheal illnesses when the cost of testing is borne by the individual, and short hospital stays. These Lao results are limited by the small sample size, the sparse clinical information available, and the use of culture alone. However, we hope that this study will alert clinicians and policy makers to the presence of this bacterium in Laos. As in many parts of Asia, this country is experiencing rapid economic and demographic changes, and changes in healthcare which will further increase the potential importance of CDI. Enhanced surveillance will be required to fully appreciate the extent and impact of CDI in Laos, and elsewhere in the region.

Conclusions
With the isolation of C.difficile from human diarrhoeal stool samples in Lao PDR, we can include this bacterium as a potential diarrhoeal pathogen in this country. This is an important first step in its recognition by clinicians and epidemiologists both within the country itself and the Greater Mekong Subregion of Asia. Future local investment in the laboratory diagnosis of CDI, the typing of isolates, and defining the clinical epidemiology of infection in the region is required to better inform healthcare providers in the development of clinical management algorithms, infection control and prevention practices, and policies for the prudent use of antimicrobials. We also wish to thank Deidre Collins and Stacey Hong for technical assistance in the typing of isolates.

Funding
The materials used for this study were made available through voluntary contributions by the Concord Hospital Microbiology trust fund. ChromID C. difficile agar plates, were kindly donated by bioMerieux, France. Typing of isolates, wages for laboratory work, manuscript review and preparation were performed free of charge. None of the entities who provided funding or voluntary contributions had a role in the study design, collection, analysis and interpretation of the data or preparation of the manuscript.

Availability of data and materials
The datasets used in this report are available from the corresponding author on reasonable request.
Authors' contributions EC conceived the protocol, completed the lab work and helped with data analysis and writing the manuscript. TR was involved in data analysis, literature review and had a primary role in manuscript writing. SR was involved in clinical data collection and data analysis. TVR was involved in subtype testing, data analysis and manuscript editing. PNN was involved in the conception of the protocol and manuscript editing. DABD was involved in the conception of the protocol, data analysis and manuscript editing. All authors have read and approved the final manuscript.
Ethics approval and consent to participate This is a report on findings from the addition of diagnostic tests being piloted in a laboratory providing routine clinical microbiology as part of hospital based clinical care. As such formal ethics approval was not requested. Patients gave oral consent for the collection of stool samples for diagnosis of their illness.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.