Antimicrobial Resistance in Invasive Non-typhoid Salmonella from the Democratic Republic of the Congo: Emergence of Decreased Fluoroquinolone Susceptibility and Extended-spectrum Beta Lactamases

Background

Co-resistance against the first-line antibiotics ampicillin, chloramphenicol and trimethoprim/sulphamethoxazole or multidrug resistance (MDR) is common in non typhoid Salmonella (NTS). Use of alternative antibiotics, such as fluoroquinolones or third generation cephalosporins is threatened by increasing resistance, but remains poorly documented in Central-Africa.

Methodology/Principal findings

As part of a microbiological surveillance study in DR Congo, blood cultures were collected between 2007 and 2011. Isolated NTS were assessed for serotype and antimicrobial resistance including decreased ciprofloxacin susceptibility and extended-spectrum beta-lactamase (ESBL) production. In total, 233 NTS isolates (representing 23.6% of clinically significant organisms) were collected, mainly consisting of Salmonella Typhimurium (79%) and Salmonella Enteritidis (18%). The majority of NTS were isolated in the rainy season, and recovered from children ≤2 years old. MDR, decreased ciprofloxacin susceptibility, azithromycin and cefotaxime resistance were 80.7%, 4.3%, 3.0% and 2.1% respectively. ESBL production was noted in three (1.3%) isolates. Decreased ciprofloxacin susceptibility was associated with mutations in codon 87 of the gyrA gene, while ESBLs all belonged to the SHV-2a type.

Conclusions/Significance

Presence of almost full MDR among NTS isolates from blood cultures in Central Africa was confirmed. Resistance to fluoroquinolones, azithromycin and third generation cephalosporins is still low, but emerging. Increased microbiological surveillance in DR Congo is crucial for adapted antibiotic therapy and the development of treatment guidelines.     

Azithromycin and Ciprofloxacin Resistance in Salmonella Bloodstream Infections in Cambodian Adults

Background

Salmonella enterica is a frequent cause of bloodstream infection (BSI) in Asia but few data are available from Cambodia. We describe Salmonella BSI isolates recovered from patients presenting at Sihanouk Hospital Centre of Hope, Phnom Penh, Cambodia (July 2007–December 2010).

Methodology

Blood was cultured as part of a microbiological prospective surveillance study. Identification of Salmonella isolates was performed by conventional methods and serotyping. Antibiotic susceptibilities were assessed using disk diffusion, MicroScan and E-test macromethod. Clonal relationships were assessed by Pulsed Field Gel Electrophoresis; PCR and sequencing for detection of mutations in Gyrase and Topoisomerase IV and presence of qnr genes.

Principal Findings

Seventy-two Salmonella isolates grew from 58 patients (mean age 34.2 years, range 8–71). Twenty isolates were identified as Salmonella Typhi, 2 as Salmonella Paratyphi A, 37 as Salmonella Choleraesuis and 13 as other non-typhoid Salmonella spp. Infection with human immunodeficiency virus (HIV) was present in 21 of 24 (87.5%) patients with S. Choleraesuis BSI. Five patients (8.7%) had at least one recurrent infection, all with S. Choleraesuis; five patients died. Overall, multi drug resistance (i.e., co-resistance to ampicillin, sulphamethoxazole-trimethoprim and chloramphenicol) was high (42/59 isolates, 71.2%). S. Typhi displayed high rates of decreased ciprofloxacin susceptibility (18/20 isolates, 90.0%), while azithromycin resistance was very common in S. Choleraesuis (17/24 isolates, 70.8%). Two S. Choleraesuis isolates were extended spectrum beta-lactamase producer.

Conclusions and Significance

Resistance rates in Salmonella spp. in Cambodia are alarming, in particular for azithromycin and ciprofloxacin. This warrants nationwide surveillance and revision of treatment guidelines.     

Bloodstream Infection among Adults in Phnom Penh,Cambodia: Key Pathogens and Resistance Patterns

Background

Bloodstream infections (BSI) cause important morbidity and mortality worldwide. In Cambodia, no surveillance data on BSI are available so far.

Methods

From all adults presenting with SIRS at Sihanouk Hospital Centre of HOPE (July 2007–December 2010), 20 ml blood was cultured. Isolates were identified using standard microbiological techniques; antibiotic susceptibilities were assessed using disk diffusion and MicroScan®, with additional E-test, D-test and double disk test where applicable, according to CLSI guidelines.

Results

A total of 5714 samples from 4833 adult patients yielded 501 clinically significant organisms (8.8%) of which 445 available for further analysis. The patients’ median age was 45 years (range 15–99 y), 52.7% were women. HIV-infection and diabetes were present in 15.6% and 8.8% of patients respectively. The overall mortality was 22.5%. Key pathogens included Escherichia coli (n = 132; 29.7%), Salmonella spp. (n = 64; 14.4%), Burkholderia pseudomallei (n = 56; 12.6%) and Staphylococcus aureus (n = 53; 11.9%). Methicillin resistance was seen in 10/46 (21.7%) S. aureus; 4 of them were co-resistant to erythromycin, clindamycin, moxifloxacin and sulphamethoxazole-trimethoprim (SMX-TMP). We noted combined resistance to amoxicillin, SMX-TMP and ciprofloxacin in 81 E. coli isolates (62.3%); 62 isolates (47.7%) were confirmed as producers of extended spectrum beta-lactamase. Salmonella isolates displayed high rates of multidrug resistance (71.2%) with high rates of decreased ciprofloxacin susceptibility (90.0%) in Salmonella Typhi while carbapenem resistance was observed in 5.0% of 20 Acinetobacter sp. isolates.

Conclusions

BSI in Cambodian adults is mainly caused by difficult-to-treat pathogens. These data urge for microbiological capacity building, nationwide surveillance and solid interventions to contain antibiotic resistance.     

High-resolution genotyping of the endemic Salmonella Typhi population during a Vi (typhoid) vaccination trial in Kolkata

Background

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a major health problem especially in developing countries. Vaccines against typhoid are commonly used by travelers but less so by residents of endemic areas.

Methodology

We used single nucleotide polymorphism (SNP) typing to investigate the population structure of 372 S. Typhi isolated during a typhoid disease burden study and Vi vaccine trial in Kolkata, India. Approximately sixty thousand people were enrolled for fever surveillance for 19 months prior to, and 24 months following, Vi vaccination of one third of the study population (May 2003–December 2006, vaccinations given December 2004).

Principal Findings

A diverse S. Typhi population was detected, including 21 haplotypes. The most common were of the H58 haplogroup (69%), which included all multidrug resistant isolates (defined as resistance to chloramphenicol, ampicillin and co-trimoxazole). Quinolone resistance was particularly high among H58-G isolates (97% Nalidixic acid resistant, 30% with reduced susceptibility to ciprofloxacin). Multiple typhoid fever episodes were detected in 22 households, however household clustering was not associated with specific S. Typhi haplotypes.

Conclusions

Typhoid fever in Kolkata is caused by a diverse population of S. Typhi, however H58 haplotypes dominate and are associated with multidrug and quinolone resistance. Vi vaccination did not obviously impact on the haplotype population structure of the S. Typhi circulating during the study period.     

Microbial-resistant Salmonella enteritidis isolated from poultry samples

Background:

Multidrug resistance in Salmonella enteritidis isolates is a public health problem worldwide; the present study, therefore, was designed for antimicrobial-resistance determination in this strain.

Methods:

Salmonella strains isolated from poultry samples by biochemical positive and negative tests were subjected to PCR and identified as Salmonella enteritidis. For detection and identification of Salmonella enteritidis isolates, sdfI gene-specific primers were used.

Results:

We found that 100% of isolates were resistant to ampicillin, 90% were resistant to cephalothin and streptomycin, 70% were resistant to cefotaxime, and 60% were resistant to kanamycin and gentamicin.

Conclusion:

Salmonella enteritidis isolates had antimicrobial resistance to mentioned antibiotics. Key Words: Antibiotic Resistance, PCR, Poultry, Salmonella enteritidis     

Genomic Characterisation of Invasive Non-Typhoidal Salmonella enterica Subspecies enterica Serovar Bovismorbificans Isolates from Malawi

Background

Invasive Non-typhoidal Salmonella (iNTS) are an important cause of bacteraemia in children and HIV-infected adults in sub-Saharan Africa. Previous research has shown that iNTS strains exhibit a pattern of gene loss that resembles that of host adapted serovars such as Salmonella Typhi and Paratyphi A. Salmonella enterica serovar Bovismorbificans was a common serovar in Malawi between 1997 and 2004.

Methodology

We sequenced the genomes of 14 Malawian bacteraemia and four veterinary isolates from the UK, to identify genomic variations and signs of host adaptation in the Malawian strains.

Principal Findings

Whole genome phylogeny of invasive and veterinary S. Bovismorbificans isolates showed that the isolates are highly related, belonging to the most common international S. Bovismorbificans Sequence Type, ST142, in contrast to the findings for S. Typhimurium, where a distinct Sequence Type, ST313, is associated with invasive disease in sub-Saharan Africa. Although genome degradation through pseudogene formation was observed in ST142 isolates, there were no clear overlaps with the patterns of gene loss seen in iNTS ST313 isolates previously described from Malawi, and no clear distinction between S. Bovismorbificans isolates from Malawi and the UK.The only defining differences between S. Bovismorbificans bacteraemia and veterinary isolates were prophage-related regions and the carriage of a S. Bovismorbificans virulence plasmid (pVIRBov).

Conclusions

iNTS S. Bovismorbificans isolates, unlike iNTS S. Typhiumrium isolates, are only distinguished from those circulating elsewhere by differences in the mobile genome. It is likely that these strains have entered a susceptible population and are able to take advantage of this niche. There are tentative signs of convergent evolution to a more human adapted iNTS variant. Considering its importance in causing disease in this region, S. Bovismorbificans may be at the beginning of this process, providing a reference against which to compare changes that may become fixed in future lineages in sub-Saharan Africa.     

CRISPR Is an Optimal Target for the Design of Specific PCR Assays for Salmonella enterica Serotypes Typhi and Paratyphi A

Background

Serotype-specific PCR assays targeting Salmonella enterica serotypes Typhi and Paratyphi A, the causal agents of typhoid and paratyphoid fevers, are required to accelerate formal diagnosis and to overcome the lack of typing sera and, in some situations, the need for culture. However, the sensitivity and specificity of such assays must be demonstrated on large collections of strains representative of the targeted serotypes and all other bacterial populations producing similar clinical symptoms.

Methodology

Using a new family of repeated DNA sequences, CRISPR (clustered regularly interspaced short palindromic repeats), as a serotype-specific target, we developed a conventional multiplex PCR assay for the detection and differentiation of serotypes Typhi and Paratyphi A from cultured isolates. We also developed EvaGreen-based real-time singleplex PCR assays with the same two sets of primers.

Principal findings

We achieved 100% sensitivity and specificity for each protocol after validation of the assays on 188 serotype Typhi and 74 serotype Paratyphi A strains from diverse genetic groups, geographic origins and time periods and on 70 strains of bacteria frequently encountered in bloodstream infections, including 29 other Salmonella serotypes and 42 strains from 38 other bacterial species.

Conclusions

The performance and convenience of our serotype-specific PCR assays should facilitate the rapid and accurate identification of these two major serotypes in a large range of clinical and public health laboratories with access to PCR technology. These assays were developed for use with DNA from cultured isolates, but with modifications to the assay, the CRISPR targets could be used in the development of assays for use with clinical and other samples.     

The burden of invasive bacterial infections in Pemba, Zanzibar

Background

We conducted a surveillance study to determine the leading causes of bloodstream infection in febrile patients seeking treatment at three district hospitals in Pemba Island, Zanzibar, Tanzania, an area with low malaria transmission.

Methods

All patients above two months of age presenting to hospital with fever were screened, and blood was collected for microbiologic culture and malaria testing. Bacterial sepsis and malaria crude incidence rates were calculated for a one-year period and were adjusted for study participation and diagnostic sensitivity of blood culture.

Results

Blood culture was performed on 2,209 patients. Among them, 166 (8%) samples yielded bacterial growth; 87 (4%) were considered as likely contaminants; and 79 (4%) as pathogenic bacteria. The most frequent pathogenic bacteria isolated were Salmonella Typhi (n = 46; 58%), followed by Streptococcus pneumoniae (n = 12; 15%). The crude bacteremia rate was 6/100,000 but when adjusted for potentially missed cases the rate may be as high as 163/100,000. Crude and adjusted rates for S. Typhi infections and malaria were 4 and 110/100,000 and 4 and 47/100,000, respectively. Twenty three (51%), 22 (49%) and 22 (49%) of the S.Typhi isolates were found to be resistant toward ampicillin, chloramphenicol and cotrimoxazole, respectively. Multidrug resistance (MDR) against the three antimicrobials was detected in 42% of the isolates.

Conclusions

In the presence of very low malaria incidence we found high rates of S. Typhi and S. pneumoniae infections on Pemba Island, Zanzibar. Preventive measures such as vaccination could reduce the febrile disease burden.     

Antibiotic Prescribing in DR Congo: A Knowledge,Attitude and Practice Survey among Medical Doctors and Students

Objectives

Antibiotic resistance (ABR) particularly hits resource poor countries, and is fuelled by irrational antibiotic (AB) prescribing. We surveyed knowledge, attitudes and practices of AB prescribing among medical students and doctors in Kisangani, DR Congo.

Methods

Self-administered questionnaires.

Results

A total of 184 questionnaires were completed (response rate 94.4%). Knowledge about AB was low (mean score 4.9/8 points), as was the estimation of local resistance rates of S. Typhi and Klebsiella spp.(correct by 42.5% and 6.9% of respondents respectively). ABR was recognized as a problem though less in their own practice (67.4%) than nation- or worldwide (92.9% and 85.5%, p<.0001). Confidence in AB prescribing was high (88.6%) and students consulted more frequently colleagues than medical doctors when prescribing (25.4% versus 11.6%, p  = 0.19). Sources of AB prescribing included pharmaceutical companies (73.9%), antibiotic guidelines (66.3%), university courses (63.6%), internet-sites (45.7%) and WHO guidelines (26.6%). Only 30.4% and 16.3% respondents perceived AB procured through the central procurement and local pharmacies as of good quality. Local AB guidelines and courses about AB prescribing are welcomed (73.4% and 98.8% respectively).

Conclusions

This data shows the need for interventions that support rational AB prescribing.     

Identification by PCR of Non-typhoidal Salmonella enterica Serovars Associated with Invasive Infections among Febrile Patients in Mali

Background

In sub-Saharan Africa, non-typhoidal Salmonella (NTS) are emerging as a prominent cause of invasive disease (bacteremia and focal infections such as meningitis) in infants and young children. Importantly, including data from Mali, three serovars, Salmonella enterica serovar Typhimurium, Salmonella Enteritidis and Salmonella Dublin, account for the majority of non-typhoidal Salmonella isolated from these patients.

Methods

We have extended a previously developed series of polymerase chain reactions (PCRs) based on O serogrouping and H typing to identify Salmonella Typhimurium and variants (mostly I 4,[5],12:i:-), Salmonella Enteritidis and Salmonella Dublin. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR was used to differentiate diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium from other O serogroup B, H:i serovars. We used these PCRs to blind-test 327 Salmonella serogroup B and D isolates that were obtained from the blood cultures of febrile patients in Bamako, Mali.

Principal Findings

We have shown that when used in conjunction with our previously described O-serogrouping PCR, our PCRs are 100% sensitive and specific in identifying Salmonella Typhimurium and variants, Salmonella Enteritidis, Salmonella Dublin and Salmonella Stanleyville. When we attempted to differentiate 171 Salmonella Typhimurium (I 4,[ 5],12:i:1,2) strains from 52 monophasic Salmonella Typhimurium (I 4,[5],12:i:-) strains, we were able to correctly identify 170 of the Salmonella Typhimurium and 51 of the Salmonella I 4,[5],12:i:- strains.

Conclusion

We have described a simple yet effective PCR method to support surveillance of the incidence of invasive disease caused by NTS in developing countries.     

Outbreak of Multi-Drug Resistant Pseudomonas aeruginosa Bloodstream Infection in the Haematology Unit of a South African Academic Hospital

Objective

To describe an outbreak of multi-resistant Pseudomonas aeruginosa bloodstream infections (MRPA-BSI) that occurred in the haematology ward of a tertiary academic hospital in Cape Town, South Africa, and determine risk factors for acquisition of MRPA-BSI.

Methods

The outbreak investigation included a search for additional cases, review of patient records, environmental and staff screening, molecular typing using pulsed-field gel electrophoresis (PFGE) and Multi-locus sequencing (MLST) and a retrospective case-control study.

Results

Ten MRPA-BSI cases occurred in the haematology ward between January 2010 and January 2011. The case fatality rate was 80%. Staff screening specimens were negative for MRPA and an environmental source was not identified. PFGE showed that 9/10 isolates were related. MLST showed that 3 of these 9 isolates belonged to Sequence type (ST) 233 while the unrelated isolate belonged to ST260.

Conclusion

We have described an outbreak of MRPA-BSI occurring over an extended period of time among neutropenic haematology patients. Molecular typing confirms that the outbreak was predominantly due to a single strain. The source of the outbreak was not identified, but the outbreak appears to have been controlled following intensive infection control measures.     

Differential Epidemiology of Salmonella Typhi and Paratyphi A in Kathmandu,Nepal: A Matched Case Control Investigation in a Highly Endemic Enteric Fever Setting

Background

Enteric fever, a systemic infection caused by the bacteria Salmonella Typhi and Salmonella Paratyphi A, is endemic in Kathmandu, Nepal. Previous work identified proximity to poor quality water sources as a community-level risk for infection. Here, we sought to examine individual-level risk factors related to hygiene and sanitation to improve our understanding of the epidemiology of enteric fever in this setting.

Methodology and principal findings

A matched case-control analysis was performed through enrollment of 103 blood culture positive enteric fever patients and 294 afebrile community-based age and gender-matched controls. A detailed questionnaire was administered to both cases and controls and the association between enteric fever infection and potential exposures were examined through conditional logistic regression. Several behavioral practices were identified as protective against infection with enteric fever, including water storage and hygienic habits. Additionally, we found that exposures related to poor water and socioeconomic status are more influential in the risk of infection with S. Typhi, whereas food consumption habits and migration play more of a role in risk of S. Paratyphi A infection.

Conclusions and significance

Our work suggests that S. Typhi and S. Paratyphi A follow different routes of infection in this highly endemic setting and that sustained exposure to both serovars probably leads to the development of passive immunity. In the absence of a polyvalent vaccine against S. Typhi and S. Paratyphi A, we advocate better systems for water treatment and storage, improvements in the quality of street food, and vaccination with currently available S. Typhi vaccines.     

Antimicrobial Resistance,Class 1 Integrons,and Genomic Island 1 in Salmonella Isolates from Vietnam

Background

The objective was to investigate the phenotypic and genotypic resistance and the horizontal transfer of resistance determinants from Salmonella isolates from humans and animals in Vietnam.

Methodology/Principal Findings

The susceptibility of 297 epidemiologically unrelated non-typhoid Salmonella isolates was investigated by disk diffusion assay. The isolates were screened for the presence of class 1 integrons and Salmonella genomic island 1 by PCR. The potential for the transfer of resistance determinants was investigated by conjugation experiments. Resistance to gentamicin, kanamycin, chloramphenicol, streptomycin, trimethoprim, ampicillin, nalidixic acid, sulphonamides, and tetracycline was found in 13 to 50% of the isolates. Nine distinct integron types were detected in 28% of the isolates belonging to 11 Salmonella serovars including S. Tallahassee. Gene cassettes identified were aadA1, aadA2, aadA5, bla _PSE-1, bla _OXA-30, dfrA1, dfrA12, dfrA17, and sat, as well as open reading frames with unknown functions. Most integrons were located on conjugative plasmids, which can transfer their antimicrobial resistance determinants to Escherichia coli or Salmonella Enteritidis, or with Salmonella Genomic Island 1 or its variants. The resistance gene cluster in serovar Emek identified by PCR mapping and nucleotide sequencing contained SGI1-J3 which is integrated in SGI1 at another position than the majority of SGI1. This is the second report on the insertion of SGI1 at this position. High-level resistance to fluoroquinolones was found in 3 multiresistant S. Typhimurium isolates and was associated with mutations in the gyrA gene leading to the amino acid changes Ser83Phe and Asp87Asn.

Conclusions

Resistance was common among Vietnamese Salmonella isolates from different sources. Legislation to enforce a more prudent use of antibiotics in both human and veterinary medicine should be implemented by the authorities in Vietnam.     

Clonal differences between Non-Typhoidal Salmonella (NTS) recovered from children and animals living in close contact in the Gambia

Background

Non-Typhoidal Salmonella (NTS) is an important cause of invasive bacterial disease and associated with mortality in Africa. However, little is known about the environmental reservoirs and predominant modes of transmission. Our study aimed to study the role of domestic animals in the transmission of NTS to humans in rural area of The Gambia.

Methodology

Human NTS isolates were obtained through an active population-based case-control surveillance study designated to determine the aetiology and epidemiology of enteric infections covering 27,567 Gambian children less than five years of age in the surveillance area. Fourteen children infected with NTS were traced back to their family compounds and anal swabs collected from 210 domestic animals present in their households. Identified NTSs were serotyped and genotyped by multi-locus sequencing typing.

Principal Findings

NTS was identified from 21/210 animal sources in the households of the 14 infected children. Chickens carried NTS more frequently than sheep and goats; 66.6%, 28.6% and 4.8% respectively. The most common NTS serovars were S. Colindale in humans (21.42%) and S. Poona in animals (14.28%). MLST on the 35 NTS revealed four new alleles and 24 sequence types (ST) of which 18 (75%) STs were novel. There was no overlap in serovars or genotypes of NTS recovered from humans or animal sources in the same household.

Conclusion

Our results do not support the hypothesis that humans and animals in close contact in the same household carry genotypically similar Salmonella serovars. These findings form an important baseline for future studies of transmission of NTS in humans and animals in Africa.     

Head-to-Head Comparison of Humoral Immune Responses to Vi Capsular Polysaccharide and Salmonella Typhi Ty21a Typhoid Vaccines–A Randomized Trial

Background

The two typhoid vaccines, the parenteral Vi capsular polysaccharide and the oral live whole-cell Salmonella Typhi Ty21a vaccine, provide similar levels of protection in field trials. Sharing no antigens, they are thought to confer protection by different mechanisms. This is the first head-to-head study to compare the humoral immune responses to these two vaccines.

Methods

50 age- and gender-matched volunteers were immunized, 25 with the Vi and 25 with the Ty21a vaccine. Circulating plasmablasts reactive with whole-cell Salmonella Typhi or one of the typhoidal antigenic structures, Vi, O-9,12, and H-d antigens, were identified as antibody-secreting cells (ASC) with ELISPOT. Homing receptor (HR) expressions were determined. These results were compared with ASC in four patients with typhoid fever. Antibodies to S. Typhi lipopolysaccharides were assessed in cultures of ALS (antibodies in lymphocyte supernatants) and in serum with ELISA.

Results

In 49 out of 50 vaccinees, no typhoid-specific plasmablasts were seen before vaccination. On day 7, response to Vi antigen was mounted in 24/25 volunteers in the Vi, and none in the Ty21a group; response to S. Typhi and O-9,12 was mounted in 49/50 vaccinees; and to H-d in 3/50. The numbers of typhoid-specific plasmablasts (total of ASC to Vi, O-9,12 and H-d antigens) proved equal in the vaccination groups. The HR expressions indicated a mainly systemic homing in the Vi and intestinal in the Ty21a group, the latter resembling that in natural infection. Plasmablasts proved more sensitive than serum and ALS in assessing the immune response.

Conclusions

The typhoid-specific humoral responses to Vi and Ty21a vaccines are similar in magnitude, but differ in expected localization and antigen-specificity. The unforeseen O antigen-specific response in the Vi group is probably due to lipopolysaccharide contaminating the vaccine preparation. Only the response to Ty21a vaccine was found to imitate that in natural infection.

Trial Registration

Current Controlled Trials Ltd. c/o BioMed Central ISRCTN68125331 http://www.controlled-trials.com/ISRCTN68125331/     

Evaluation of an Electricity-free,Culture-based Approach for Detecting Typhoidal Salmonella Bacteremia during Enteric Fever in a High Burden,Resource-limited Setting

Background

In many rural areas at risk for enteric fever, there are few data on Salmonella enterica serotypes Typhi (S. Typhi) and Paratyphi (S. Paratyphi) incidence, due to limited laboratory capacity for microbiologic culture. Here, we describe an approach that permits recovery of the causative agents of enteric fever in such settings. This approach involves the use of an electricity-free incubator based upon use of phase-change materials. We compared this against conventional blood culture for detection of typhoidal Salmonella.

Methodology/Principal Findings

Three hundred and four patients with undifferentiated fever attending the outpatient and emergency departments of a public hospital in the Kathmandu Valley of Nepal were recruited. Conventional blood culture was compared against an electricity-free culture approach. Blood from 66 (21.7%) patients tested positive for a Gram-negative bacterium by at least one of the two methods. Sixty-five (21.4%) patients tested blood culture positive for S. Typhi (30; 9.9%) or S. Paratyphi A (35; 11.5%). From the 65 individuals with culture-confirmed enteric fever, 55 (84.6%) were identified by the conventional blood culture and 60 (92.3%) were identified by the experimental method. Median time-to-positivity was 2 days for both procedures. The experimental approach was falsely positive due to probable skin contaminants in 2 of 239 individuals (0.8%). The percentages of positive and negative agreement for diagnosis of enteric fever were 90.9% (95% CI: 80.0%–97.0%) and 96.0% (92.7%–98.1%), respectively. After initial incubation, Salmonella isolates could be readily recovered from blood culture bottles maintained at room temperature for six months.

Conclusions/Significance

A simple culture approach based upon a phase-change incubator can be used to isolate agents of enteric fever. This approach could be used as a surveillance tool to assess incidence and drug resistance of the etiologic agents of enteric fever in settings without reliable local access to electricity or local diagnostic microbiology laboratories.     

Dynamic Modularity of Host Protein Interaction Networks in Salmonella Typhi Infection

Background

Salmonella Typhi is a human-restricted pathogen, which causes typhoid fever and remains a global health problem in the developing countries. Although previously reported host expression datasets had identified putative biomarkers and therapeutic targets of typhoid fever, the underlying molecular mechanism of pathogenesis remains incompletely understood.

Methods

We used five gene expression datasets of human peripheral blood from patients suffering from S. Typhi or other bacteremic infections or non-infectious disease like leukemia. The expression datasets were merged into human protein interaction network (PIN) and the expression correlation between the hubs and their interacting proteins was measured by calculating Pearson Correlation Coefficient (PCC) values. The differences in the average PCC for each hub between the disease states and their respective controls were calculated for studied datasets. The individual hubs and their interactors with expression, PCC and average PCC values were treated as dynamic subnetworks. The hubs that showed unique trends of alterations specific to S. Typhi infection were identified.

Results

We identified S. Typhi infection-specific dynamic subnetworks of the host, which involve 81 hubs and 1343 interactions. The major enriched GO biological process terms in the identified subnetworks were regulation of apoptosis and biological adhesions, while the enriched pathways include cytokine signalling in the immune system and downstream TCR signalling. The dynamic nature of the hubs CCR1, IRS2 and PRKCA with their interactors was studied in detail. The difference in the dynamics of the subnetworks specific to S. Typhi infection suggests a potential molecular model of typhoid fever.

Conclusions

Hubs and their interactors of the S. Typhi infection-specific dynamic subnetworks carrying distinct PCC values compared with the non-typhoid and other disease conditions reveal new insight into the pathogenesis of S. Typhi.     

Population-based incidence of typhoid fever in an urban informal settlement and a rural area in Kenya: implications for typhoid vaccine use in Africa

Background

High rates of typhoid fever in children in urban settings in Asia have led to focus on childhood immunization in Asian cities, but not in Africa, where data, mostly from rural areas, have shown low disease incidence. We set out to compare incidence of typhoid fever in a densely populated urban slum and a rural community in Kenya, hypothesizing higher rates in the urban area, given crowding and suboptimal access to safe water, sanitation and hygiene.

Methods

During 2007-9, we conducted population-based surveillance in Kibera, an urban informal settlement in Nairobi, and in Lwak, a rural area in western Kenya. Participants had free access to study clinics; field workers visited their homes biweekly to collect information about acute illnesses. In clinic, blood cultures were processed from patients with fever or pneumonia. Crude and adjusted incidence rates were calculated.

Results

In the urban site, the overall crude incidence of Salmonella enterica serovar Typhi (S. Typhi) bacteremia was 247 cases per 100,000 person-years of observation (pyo) with highest rates in children 5–9 years old (596 per 100,000 pyo) and 2–4 years old (521 per 100,000 pyo). Crude overall incidence in Lwak was 29 cases per 100,000 pyo with low rates in children 2–4 and 5–9 years old (28 and 18 cases per 100,000 pyo, respectively). Adjusted incidence rates were highest in 2–4 year old urban children (2,243 per 100,000 pyo) which were >15-fold higher than rates in the rural site for the same age group. Nearly 75% of S. Typhi isolates were multi-drug resistant.

Conclusions

This systematic urban slum and rural comparison showed dramatically higher typhoid incidence among urban children <10 years old with rates similar to those from Asian urban slums. The findings have potential policy implications for use of typhoid vaccines in increasingly urban Africa.     

Temporal fluctuation of multidrug resistant salmonella typhi haplotypes in the mekong river delta region of Vietnam

Background

Typhoid fever remains a public health problem in Vietnam, with a significant burden in the Mekong River delta region. Typhoid fever is caused by the bacterial pathogen Salmonella enterica serovar Typhi (S. Typhi), which is frequently multidrug resistant with reduced susceptibility to fluoroquinolone-based drugs, the first choice for the treatment of typhoid fever. We used a GoldenGate (Illumina) assay to type 1,500 single nucleotide polymorphisms (SNPs) and analyse the genetic variation of S. Typhi isolated from 267 typhoid fever patients in the Mekong delta region participating in a randomized trial conducted between 2004 and 2005.

Principal Findings

The population of S. Typhi circulating during the study was highly clonal, with 91% of isolates belonging to a single clonal complex of the S. Typhi H58 haplogroup. The patterns of disease were consistent with the presence of an endemic haplotype H58-C and a localised outbreak of S. Typhi haplotype H58-E2 in 2004. H58-E2-associated typhoid fever cases exhibited evidence of significant geo-spatial clustering along the Sông H u branch of the Mekong River. Multidrug resistance was common in the established clone H58-C but not in the outbreak clone H58-E2, however all H58 S. Typhi were nalidixic acid resistant and carried a Ser83Phe amino acid substitution in the gyrA gene.

Significance

The H58 haplogroup dominates S. Typhi populations in other endemic areas, but the population described here was more homogeneous than previously examined populations, and the dominant clonal complex (H58-C, -E1, -E2) observed in this study has not been detected outside Vietnam. IncHI1 plasmid-bearing S. Typhi H58-C was endemic during the study period whilst H58-E2, which rarely carried the plasmid, was only transient, suggesting a selective advantage for the plasmid. These data add insight into the outbreak dynamics and local molecular epidemiology of S. Typhi in southern Vietnam.