Michel M. Dione

Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study

BACKGROUND Childhood diarrhoea can be caused by many pathogens that are difficult to assay in the laboratory. Molecular diagnostic techniques provide a uniform method to detect and quantify candidate enteropathogens. We aimed to develop and assess molecular tests for identification of enteropathogens and their association with disease. METHODS We developed and assessed molecular diagnostic tests for 15 enteropathogens across three platforms-PCR-Luminex, multiplex real-time PCR, and TaqMan array card-at five laboratories worldwide. We judged the analytical and clinical performance of these molecular techniques against comparator methods (bacterial culture, ELISA, and PCR) using 867 diarrhoeal and 619 non-diarrhoeal stool specimens. We also measured molecular quantities of pathogens to predict the association with diarrhoea, by univariate logistic regression analysis. FINDINGS The molecular tests showed very good analytical and clinical performance at all five laboratories. Comparator methods had limited sensitivity compared with the molecular techniques (20-85% depending on the target) but good specificity (median 97·3%, IQR 96·5-98·9; mean 95·2%, SD 9·1). Positive samples by comparator methods usually had higher molecular quantities of pathogens than did negative samples, across almost all platforms and for most pathogens (p<0·05). The odds ratio for diarrhoea at a given quantity (measured by quantification cycle, Cq) showed that for most pathogens associated with diarrhoea-including Campylobacter jejuni and Campylobacter coli, Cryptosporidium spp, enteropathogenic Escherichia coli, heat-stable enterotoxigenic E coli, rotavirus, Shigella spp and enteroinvasive E coli, and Vibrio cholerae-the strength of association with diarrhoea increased at higher pathogen loads. For example, Shigella spp at a Cq range of 15-20 had an odds ratio of 8·0 (p<0·0001), but at a Cq range of 25-30 the odds ratio fell to 1·7 (p=0·043). INTERPRETATION Molecular diagnostic tests can be implemented successfully and with fidelity across laboratories around the world. In the case of diarrhoea, these techniques can detect pathogens with high sensitivity and ascribe diarrhoeal associations based on quantification, including in mixed infections, providing rich and unprecedented measurements of infectious causes. FUNDING Bill & Melinda Gates Foundation Next Generation Molecular Diagnostics Project.

Quantitative PCR for detection of Shigella improves ascertainment of Shigella burden in children with moderate-to-severe diarrhea in low-income countries.

ABSTRACT Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 104 ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 104 ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ≥2.9 × 104 ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 104 ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture in both samples. Among a subset (n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n = 129) for culture to 17.6% (n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 104 ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella.

Factors Associated with Siman Immunodeficiency Virus Transmission in a Natural African Nonhuman Primate Host in the Wild

ABSTRACT African green monkeys (AGMs) are naturally infected with simian immunodeficiency virus (SIV) at high prevalence levels and do not progress to AIDS. Sexual transmission is the main transmission route in AGM, while mother-to-infant transmission (MTIT) is negligible. We investigated SIV transmission in wild AGMs to assess whether or not high SIV prevalence is due to differences in mucosal permissivity to SIV (i.e., whether the genetic bottleneck of viral transmission reported in humans and macaques is also observed in AGMs in the wild). We tested 121 sabaeus AGMs (Chlorocebus sabaeus) from the Gambia and found that 53 were SIV infected (44%). By combining serology and viral load quantitation, we identified 4 acutely infected AGMs, in which we assessed the diversity of the quasispecies by single-genome amplification (SGA) and documented that a single virus variant established the infections. We thus show that natural SIV transmission in the wild is associated with a genetic bottleneck similar to that described for mucosal human immunodeficiency virus (HIV) transmission in humans. Flow cytometry assessment of the immune cell populations did not identify major differences between infected and uninfected AGM. The expression of the SIV coreceptor CCR5 on CD4+ T cells dramatically increased in adults, being higher in infected than in uninfected infant and juvenile AGMs. Thus, the limited SIV MTIT in natural hosts appears to be due to low target cell availability in newborns and infants, which supports HIV MTIT prevention strategies aimed at limiting the target cells at mucosal sites. Combined, (i) the extremely high prevalence in sexually active AGMs, (ii) the very efficient SIV transmission in the wild, and (iii) the existence of a fraction of multiparous females that remain uninfected in spite of massive exposure to SIV identify wild AGMs as an acceptable model of exposed, uninfected individuals. IMPORTANCE We report an extensive analysis of the natural history of SIVagm infection in its sabaeus monkey host, the African green monkey species endemic to West Africa. Virtually no study has investigated the natural history of SIV infection in the wild. The novelty of our approach is that we report for the first time that SIV infection has no discernible impact on the major immune cell populations in natural hosts, thus confirming the nonpathogenic nature of SIV infection in the wild. We also focused on the correlates of SIV transmission, and we report, also for the first time, that SIV transmission in the wild is characterized by a major genetic bottleneck, similar to that described for HIV-1 transmission in humans. Finally, we report here that the restriction of target cell availability is a major correlate of the lack of SIV transmission to the offspring in natural hosts of SIVs.

The genome of the vervet (Chlorocebus aethiops sabaeus)

We describe a genome reference of the African green monkey or vervet (Chlorocebus aethiops). This member of the Old World monkey (OWM) superfamily is uniquely valuable for genetic investigations of simian immunodeficiency virus (SIV), for which it is the most abundant natural host species, and of a wide range of health-related phenotypes assessed in Caribbean vervets (C. a. sabaeus), whose numbers have expanded dramatically since Europeans introduced small numbers of their ancestors from West Africa during the colonial era. We use the reference to characterize the genomic relationship between vervets and other primates, the intra-generic phylogeny of vervet subspecies, and genome-wide structural variations of a pedigreed C. a. sabaeus population. Through comparative analyses with human and rhesus macaque, we characterize at high resolution the unique chromosomal fission events that differentiate the vervets and their close relatives from most other catarrhine primates, in whom karyotype is highly conserved. We also provide a summary of transposable elements and contrast these with the rhesus macaque and human. Analysis of sequenced genomes representing each of the main vervet subspecies supports previously hypothesized relationships between these populations, which range across most of sub-Saharan Africa, while uncovering high levels of genetic diversity within each. Sequence-based analyses of major histocompatibility complex (MHC) polymorphisms reveal extremely low diversity in Caribbean C. a. sabaeus vervets, compared to vervets from putatively ancestral West African regions. In the C. a. sabaeus research population, we discover the first structural variations that are, in some cases, predicted to have a deleterious effect; future studies will determine the phenotypic impact of these variations.

Risk factors, perceptions and practices associated with Taenia solium cysticercosis and its control in the smallholder pig production systems in Uganda: a cross-sectional survey.

BackgroundPrevalence studies report Taenia solium cysticercosis in pig and human populations in Uganda. However, the factors influencing occurrence in smallholder pig production systems are not well documented and little is known about farmers’ perceptions of T. solium cysticercosis or farmer practices that could reduce transmission.MethodsTo determine the risk factors, perceptions and practices regarding T. solium cysticercosis, a household survey using a semi-structured questionnaire was conducted in 1185 households in the rural and urban pig production systems in Masaka, Mukono and Kamuli Districts. Logistic regression was used to measure associations of risk factors with infection. Performance scores were calculated to summarise perceptions and practices of farmers regarding taeniosis, human cysticercosis and porcine cysticercosis as well as farmer behavior related to control or breaking transmission.ResultsPig breed type, farmers’ knowledge about transmission, sources of water used, and pig keeping homes where family members were unable to use the latrine were all significantly associated with T. solium cysticercosis in pigs. Performance scores indicated that farmers were more aware of taeniosis (63.0%; 95% Confidence Interval 60.0-65.8) than human or porcine cysticercosis; only three farmers (0.3%, 95% CI = 0.1–0.8) had knowledge on all three conditions. More farmers reported that they dewormed pigs (94.1%) than reported deworming themselves and their family members (62.0%). Albendazole was the most commonly used drug for deworming both pigs and humans (85.0 and 81.5% respectively). Just over half (54.6%) of the farmers interviewed had clean water near the latrines for washing hands. Of these, only 41.9% used water with soap to wash hands after latrine use.ConclusionFactors that significantly influenced occurrence of T. solium cysticercosis in pigs were identified. Farmers had some knowledge about the disease but did not link taeniosis, human cysticercosis, and porcine cysticercosis. Therefore, there is need to employ strategies that raise awareness and interrupt transmission.

Etiology of Severe Childhood Pneumonia in The Gambia, West Africa, Determined by Conventional and Molecular Microbiological Analyses of Lung and Pleural Aspirate Samples

Molecular analyses of lung aspirates from Gambian children with severe pneumonia detected pathogens more frequently than did culture and showed a predominance of bacteria, principally Streptococcuspneumoniae, >75% being of serotypes covered by current pneumococcal conjugate vaccines. Multiple pathogens were detected frequently, notably Haemophilus influenzae (mostly nontypeable) together with S. pneumoniae.

Participatory assessment of animal health and husbandry practices in smallholder pig production systems in three high poverty districts in Uganda

While animal health constraints have been identified as a major limiting factor in smallholder pig production in Uganda, researchers and policy makers lack information on the relative incidence of diseases and their impacts on pig production. This study aimed to assess animal health and management practices, constraints and opportunities for intervention in smallholder pig value chains in three high poverty districts of Uganda. Semi-qualitative interview checklists through Focus Group Discussions (FGDs) were administered to 340 pig farmers in 35 villages in Masaka, Kamuli and Mukono districts. Quantitative data was obtained during the exercise through group consensus. Results of FGDs were further triangulated with secondary data and information obtained from key informant interviews. Findings show that pig keeping systems are dominated by tethering and scavenging in rural areas. In peri-urban and urban areas, intensive production systems are more practiced, with pigs confined in pens. The main constraints identified by farmers include high disease burden such as African swine fever (ASF) and parasites, poor housing and feeding practices, poor veterinary services, ineffective drugs and a general lack of knowledge on piggery management. According to farmers, ASF is the primary cause of pig mortality with epidemics occurring mainly during the dry season. Worms and ectoparasites namely; mange, lice and flies are endemic leading to stunted growth which reduces the market value of pigs. Diarrhoea and malnutrition are common in piglets. Ninety-three percent of farmers say they practice deworming, 37% practice ectoparasite spraying and 77% castrate their boars. Indigenous curative treatments include the application of human urine and concoctions of local herbs for ASF control and use of old engine oil or tobacco extracts to control ectoparasites. There is a need for better technical services to assist farmers with these problems.

Prevalence and antimicrobial resistance of Salmonella isolated from broiler farms, chicken carcasses, and street-vended restaurants in Casamance, Senegal.

This study was undertaken to determine the prevalence and distribution of Salmonella on 57 randomly selected broiler farms at the end of the rearing period and in chicken products in urban and periurban areas in Casamance, Senegal, and to evaluate the antimicrobial resistance profiles of the Salmonella serovars. Salmonella was detected in chicken feces, on carcass skin, and in muscle on 35.1, 38.6, and 29.8% of farms, respectively. Salmonella was found in chicken meat servings from 14.3% of the 42 street restaurants and in 40.4% of the 285 chicken carcasses examined. The prevalence on skin and in muscle was significantly associated with the detection of Salmonella in feces (P <or= 0.001). Eighteen Salmonella serovars were identified; the most common were Brancaster (57.9%), Goelzau (10.7%), Kentucky (8.4%), and Hadar (7.3%). High levels of resistance were found to trimethoprim-sulfamethoxazole, tetracycline, trimethoprim, streptomycin, and sulfonamides. All Salmonella serovars were susceptible to fluoroquinolones and third-generation cephalosporins. A large proportion of the isolates belonging to 11 serovars were resistant to two or more antibiotics. Salmonella continues to be of serious concern in the broiler production chain in Senegal.

Seroprevalence of Leishmania infantum in a rural area of Senegal: analysis of risk factors involved in transmission to humans

Whereas Leishmania infantum, the agent of visceral leishmaniasis (VL), is well known in North Africa, very limited data exist on its spread in West Africa, where mainly cutaneous leishmaniasis has been widely reported. Nevertheless, dogs infected with L. infantum were recently found in the Mont Rolland District in Senegal. To provide a better understanding of L. infantum epidemiology in this area, clinical and serological surveys were carried out to determine the seroprevalence of L. infantum-specific antibodies in the human population. In parallel, an analysis of environmental and individual factors associated with Leishmania antigen seropositivity was conducted to identify potential risk factors for exposure. Although no cases of VL were detected within this study, a large part of the population (73/315; 23%) was exposed to infection, with a strong age effect (being >40 years old increased the risk of being seropositive). Moreover, the presence of Nebedaye trees (Moringa oleifera) and infected dogs in the household were factors increasing the risk of exposure in household members. These results may provide important information to identify the still unknown sandfly species involved in transmission.

Survey of Culture, GoldenGate Assay, Universal Biosensor Assay, and 16S rRNA Gene Sequencing as Alternative Methods of Bacterial Pathogen Detection

ABSTRACT Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens.