Prevalence, numbers and antimicrobial susceptibilities of Salmonella serovars and Campylobacter spp. in retail poultry in Phnom Penh, Cambodia.

Salmonella and Campylobacter are common bacterial pathogens associated with human gastro-enteritis; and raw poultry is considered to be an important source of these bacteria. To evaluate whether the Salmonella serovars and Campylobacter spp. bacteria could be monitored for the purpose of microbial presence, enumeration and antimicrobial resistance in raw poultry, 152 poultry carcasses were randomly selected from 10 markets in retail outlets of Phnom Penh during March 2006 to February 2007. The majority of poultry samples was contaminated by Salmonella serovars (88.2%) and Campylobacter spp. (80.9%). A very high contamination of Salmonella was found at 3-4 log10 CFU/g for 22.4% of samples and of Campylobacter at 7-8 log10 CFU/g for 1.3% of samples. Fifty nine different Salmonella serovars contaminated 134 poultry carcasses; five most prevalent serovars covered 29.1% of serovars isolates (Anatum, Typhimurium, Corvallis, Stanley and Enteritidis). Three Campylobacter species contaminating 123 raw poultry were Campylobacter jejuni (50.0%), Campylobacter coli (29.0%) and Campylobacter lari (21.0%). High antibiotic resistance percentages were found among Salmonella serovars and Campylobacter spp. isolates. This study revealed that raw poultry at the retail outlets in Phnom Penh markets are contaminated with high prevalences of food-borne pathogens, and communicating the importance of minimizing this risk in reducing human infections.

Comparisons between geographically diverse samples of carried Staphylococcus aureus.

Approximately one-third of the human population is asymptomatically colonized by Staphylococcus aureus. However, much of the global diversity within the carriage populations remains uncharacterized, and it is unclear to what degree the variation is geographically partitioned. We isolated 300 carriage isolates from 1,531 adults contemporaneously in four countries: France, Algeria, Moldova, and Cambodia. All strains were characterized by multilocus sequence typing. Six clonal complexes (CCs) were present in all four samples (CC30, -45, -121, -15, -5, and -8). Analyses based on the genotype frequencies revealed the French and Algerian samples to be most similar and the Cambodian sample to be most distinct. While this pattern is consistent with likely rates of human migration and geographic distance, stochastic clonal expansion also contributes to regional differences. Phylogenetic analysis revealed a highly divergent and uncharacterized genotype (ST1223) within Cambodia. This lineage is related to CC75, which has previously been observed only in remote aboriginal populations in northern Australia.

CTX-M beta-lactamases in Escherichia coli from community-acquired urinary tract infections, Cambodia.

Despite the recent global spread of CTX-M beta-lactamases in Escherichia coli isolates from community-acquired urinary tract infections (CA-UTIs), their dissemination has been little studied in developing countries. In a 2-year prospective study, we documented the prevalence of extended-spectrum beta-lactamases (ESBLs) in E. coli that were responsible for CA-UTIs in Phnom-Penh, Cambodia. Ninety-three E. coli strains were included. We observed a high prevalence of resistance to amoxicillin (88.2% of strains), cotrimoxazole (75.3%), ciprofloxacin (67.7%), gentamicin (42.5%), and third-generation cephalosporins (37.7%). A total of 34 strains carried ESBLs, all of which were CTX-M type. CTX-M carriage was associated with resistance to fluoroquinolones and aminoglycosides. U using repetitive extragenic palindromic-PCR, we identified 4 clusters containing 9, 8, 3, and 2 strains. The prevalence of CTX-M beta-lactamases has reached a critical level in Cambodia, which highlights the need for study of their spread in developing countries.

Diversity of staphylococcal cassette chromosome mec structures in methicillin-resistant Staphylococcus epidermidis and Staphylococcus haemolyticus strains among outpatients from four countries.

In staphylococci, methicillin (meticillin) resistance (MR) is mediated by the acquisition of the mecA gene, which is carried on the size and composition variable staphylococcal cassette chromosome mec (SCCmec). MR has been extensively studied in Staphylococcus aureus, but little is known about MR coagulase-negative staphylococci (MR-CoNS). Here, we describe the diversity of SCCmec structures in MR-CoNS from outpatients living in countries with contrasting environments: Algeria, Mali, Moldova, and Cambodia. Their MR-CoNS nasal carriage rates were 29, 17, 11, and 31%, respectively. Ninety-six MR-CoNS strains, comprising 75 (78%) Staphylococcus epidermidis strains, 19 (20%) Staphylococcus haemolyticus strains, 1 (1%) Staphylococcus hominis strain, and 1 (1%) Staphylococcus cohnii strain, were analyzed. Eighteen different SCCmec types were observed, with 28 identified as type IV (29%), 25 as type V (26%), and 1 as type III (1%). Fifteen strains (44%) were untypeable for their SCCmec. Thirty-four percent of MR-CoNS strains contained multiple ccr copies. Type IV and V SCCmec were preferentially associated with S. epidermidis and S. haemolyticus, respectively. MR-CoNS constitute a widespread and highly diversified MR reservoir in the community.

Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains.

OBJECTIVES: Recently, a CTX-M-15 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli O25b-ST131 clone, belonging to the B2 phylogenetic group and with a high virulence potential, has been reported all over the world, representing a major public health problem. The present study was carried out to develop a rapid and simple detection assay that identifies members of this clone. METHODS: A total of 627 E. coli isolates of which 373 produced an ESBL, collected across four continents, were screened using a O25b-ST131 clone allele-specific PCR for the pabB gene. RESULTS: One hundred and forty-three ESBL isolates were found positive with the assay. These isolates were all of O25b type and, when studied by multilocus sequence typing (25 cases), were all of ST131. The O25b-ST131 clone was found to produce ESBLs other than CTX-M-15, specifically CTX-M-2, -3, -14, -27, -32 and -61 as well as TEM-24. This clone represents 3% of non-ESBL B2 isolates originating from urinary tract infections in Paris. CONCLUSIONS: We have developed a PCR-based assay that easily identifies a clone with high likelihood of producing ESBLs, including CTX-M-15.

Anti-tuberculosis drug resistance and HIV co-infection in Phnom Penh, Cambodia.

The objective of this study was to observe the prevalence of drug resistance in Mycobacterium tuberculosis isolates in HIV associated tuberculosis co-infected patients in Phnom Penh City. The isolates of M. tuberculosis were collected during active laboratory-based surveillance. Of the 98 isolates studied, M. tuberculosis resistance to isoniazid was seen in 23.5%, resistance to rifampicin was seen in 16.3% and multidrug-resistance (MDR-TB) was seen in 5.1%. Our findings reveal an alarmingly high level of resistance to isoniazid and rifampicin, and confirms the need for drug susceptibility testing to guide treatment in patients with culture positive tuberculosis.

The rise and fall of malaria in a West African rural community, Dielmo, Senegal, from 1990 to 2012: a 22 year longitudinal study.

BACKGROUND: A better understanding of the effect of malaria control interventions on vector and parasite populations, acquired immunity, and burden of the disease is needed to guide strategies to eliminate malaria from highly endemic areas. We monitored and analysed the changes in malaria epidemiology in a village community in Senegal, west Africa, over 22 years. METHODS: Between 1990 and 2012, we did a prospective longitudinal study of the inhabitants of Dielmo, Senegal, to identify all episodes of fever and investigate the relation between malaria host, vector, and parasite. Our study included daily medical surveillance with systematic parasite detection in individuals with fever. We measured parasite prevalence four times a year with cross-sectional surveys. We monitored malaria transmission monthly with night collection of mosquitoes. Malaria treatment changed over the years, from quinine (1990-94), to chloroquine (1995-2003), amodiaquine plus sulfadoxine-pyrimethamine (2003-06), and finally artesunate plus amodiaquine (2006-12). Insecticide-treated nets (ITNs) were introduced in 2008. FINDINGS: We monitored 776 villagers aged 0-101 years for 2 378 150 person-days of follow-up. Entomological inoculation rate ranged from 142·5 infected bites per person per year in 1990 to 482·6 in 2000, and 7·6 in 2012. Parasite prevalence in children declined from 87% in 1990 to 0·3 % in 2012. In adults, it declined from 58% to 0·3%. We recorded 23 546 fever episodes during the study, including 8243 clinical attacks caused by Plasmodium falciparum, 290 by Plasmodium malariae, and 219 by Plasmodium ovale. Three deaths were directly attributable to malaria, and two to severe adverse events of antimalarial drugs. The incidence of malaria attacks ranged from 1·50 attacks per person-year in 1990 to 2·63 in 2000, and to only 0·046 in 2012. The greatest changes were associated with the replacement of chloroquine and the introduction of ITNs. INTERPRETATION: Malaria control policies combining prompt treatment of clinical attacks and deployment of ITNs can nearly eliminate parasite carriage and greatly reduce the burden of malaria in populations exposed to intense perennial malaria transmission. The choice of drugs seems crucial. Rapid decline of clinical immunity allows rapid detection and treatment of novel infections and thus has a key role in sustaining effectiveness of combining artemisinin-based combination therapy and ITNs despite increasing pyrethroid resistance. FUNDING: Pasteur Institutes of Dakar and Paris, Institut de Recherche pour le Développement, and French Ministry of Cooperation.

MLVA polymorphism of Salmonella enterica subspecies isolated from humans, animals, and food in Cambodia.

BACKGROUND: Salmonella (S.) enterica is the main cause of salmonellosis in humans and animals. The epidemiology of this infection involves large geographical distances, and strains related to an episode of salmonellosis therefore need to be reliably discriminated. Due to the limitations of serotyping, molecular genotyping methods have been developed, including multiple loci variable number of tandem repeats (VNTR) analysis (MLVA). In our study, 11 variable number tandem-repeats markers were selected from the S. enterica Typhimurium LT2 genome to evaluate the genetic diversity of 206 S. enterica strains collected in Cambodia between 2001 and 2007. FINDINGS: Thirty one serovars were identified from three sources: humans, animals and food. The markers were able to discriminate all strains from 2 to 17 alleles. Using the genotype phylogeny repartition, MLVA distinguished 107 genotypes clustered into two main groups: S. enterica Typhi and other serovars. Four serovars (Derby, Schwarzengrund, Stanley, and Weltevreden) were dispersed in 2 to 5 phylogenic branches. Allelic variations within S. enterica serovars was represented using the minimum spanning tree. For several genotypes, we identified clonal complexes within the serovars. This finding supports the notion of endemo-epidemic diffusion within animals, food, or humans. Furthermore, a clonal transmission from one source to another was reported. Four markers (STTR3, STTR5, STTR8, and Sal20) presented a high diversity index (DI > 0.80). CONCLUSIONS: In summary, MLVA can be used in the typing and genetic profiling of a large diversity of S. enterica serovars, as well as determining the epidemiological relationships of the strains with the geography of the area.

In vitro antifungal-drug susceptibilities of mycelial and yeast forms of Penicillium marneffei isolates in Cambodia.

Penicillium marneffei infection is an important disease among human immunodeficiency virus patients in Southeast Asia. The in vitro antifungal-drug susceptibilities of 29 clinical isolates and 5 isolates from bamboo rats collected from 2002 to 2004 were determined. The P. marneffei yeast form is more susceptible than the mycelial form to amphotericin B and ketoconazole, while the mycelial and yeast forms displayed similar susceptibilities to flucytosine and itraconazole. The MICs of fluconazole were higher for both mycelial and yeast forms.

Nipah virus in Lyle's flying foxes, Cambodia.

We conducted a survey in Cambodia in 2000 on henipavirus infection among several bat species, including flying foxes, and persons exposed to these animals. Among 1,072 bat serum samples tested by enzyme-linked immunosorbent assay, antibodies reactive to Nipah virus (NiV) antigen were detected only in Pteropus lylei species; Cynopterus sphinx, Hipposideros larvatus, Scotophilus kuhlii, Chaerephon plicata, Taphozous melanopogon, and T. theobaldi species were negative. Seroneutralization applied on a subset of 156 serum samples confirmed these results. None of the 8 human serum samples was NiV seropositive with the seroneutralization test. One virus isolate exhibiting cytopathic effect with syncytia was obtained from 769 urine samples collected at roosts of P. lylei specimens. Partial molecular characterization of this isolate demonstrated that it was closely related to NiV. These results strengthen the hypothesis that flying foxes could be the natural host of NiV. Surveillance of human cases should be implemented.

Increasing in vitro resistance to fluconazole in Cryptococcus neoformans Cambodian isolates: April 2000 to March 2002.

OBJECTIVES: Cryptococcal meningitis is the third-most-common opportunistic infection in HIV patients in Cambodia. Hospitalized patients were given amphotericin B for initial therapy followed by fluconazole for maintenance therapy. The antifungal drug susceptibility of Cryptococcus neoformans isolated from cerebrospinal fluid (CSF) was determined. METHODS: Isolates of C. neoformans were collected during active laboratory-based surveillance, the first batch from April 2000 to March 2001 (134 new cases), the second batch from April 2001 to March 2002 (268 new cases). Etest strips were used to determine the MICs of amphotericin B and fluconazole. The antigenic agglutination slide test was used for serotyping. RESULTS: The MIC(50)s and MIC(90)s of fluconazole changed significantly from year 2000 to 2002; the MIC(50)s increased from 4 to 12 mg/L, and the MIC(90)s from 12 to 96 mg/L. For amphotericin B, the MIC(50)s and MIC(90)s remained stable. Moreover, in the second batch, fluconazole MICs were >/=256 mg/L for 20 isolates. By serotyping, it was found that 98.5% of the isolates were serotype A. CONCLUSIONS: C. neoformans strains isolated from CSF of AIDS patients in Cambodia remain susceptible in vitro to amphotericin B. These strains are less susceptible in vitro to fluconazole, 2.5% being resistant in the first year and 14% in the second year of study. Nevertheless, in vitro resistance of C. neoformans to fluconazole appeared to be linked to extended maintenance treatments.