Stress response regulators identified through genome-wide transcriptome analysis of the (p)ppGpp-dependent response in Rhizobium etli

Background: The alarmone (p) ppGpp mediates a global reprogramming of gene expression upon nutrient limitation and other stresses to cope with these unfavorable conditions. Synthesis of (p) ppGpp is, in most bacteria, controlled by RelA/SpoT (Rsh) proteins. The role of (p) ppGpp has been characterized primarily in Escherichia coli and several Gram-positive bacteria. Here, we report the first in-depth analysis of the (p) ppGpp-regulon in an alpha-proteobacterium using a high-resolution tiling array to better understand the pleiotropic stress phenotype of a relA/rsh mutant. Results: We compared gene expression of the Rhizobium etli wild type and rsh (previously rel) mutant during exponential and stationary phase, identifying numerous (p) ppGpp targets, including small non-coding RNAs. The majority of the 834 (p) ppGpp-dependent genes were detected during stationary phase. Unexpectedly, 223 genes were expressed (p) ppGpp-dependently during early exponential phase, indicating the hitherto unrecognized importance of (p) ppGpp during active growth. Furthermore, we identified two (p) ppGpp-dependent key regulators for survival during heat and oxidative stress and one regulator putatively involved in metabolic adaptation, namely extracytoplasmic function sigma factor EcfG2/PF00052, transcription factor CH00371, and serine protein kinase PrkA. Conclusions: The regulatory role of (p) ppGpp in R. etli stress adaptation is far-reaching in redirecting gene expression during all growth phases. Genome-wide transcriptome analysis of a strain deficient in a global regulator, and exhibiting a pleiotropic phenotype, enables the identification of more specific regulators that control genes associated with a subset of stress phenotypes. This work is an important step toward a full understanding of the regulatory network underlying stress responses in alpha-proteobacteria

Visceral Leishmaniasis-HIV Coinfection as a Predictor of Increased Leishmania Transmission at the Village Level in Bihar, India.

BACKGROUND: Visceral leishmaniasis (VL) is on the verge of being eliminated as a public health problem in the Indian subcontinent. Although Post-kala-azar dermal leishmaniasis (PKDL) is recognized as an important reservoir of transmission, we hypothesized that VL patients co-infected with Human Immunodeficiency Virus (HIV) may also be important reservoirs of sustained leishmania transmission. We therefore investigated to what extent cases of PKDL or VL-HIV are associated with VL incidence at the village level in Bihar, India. METHODS: VL, VL-HIV, and PKDL case data from six districts within the highly VL-endemic state of Bihar, India were collected through the Kala-Azar Management Information System for the years 2014-2019. Multivariate analysis was done using negative binomial regression controlling for year as a fixed effect and block (subdistrict) as a random effect. FINDINGS: Presence of VL-HIV+ and PKDL cases were both associated with a more than twofold increase in VL incidence at village level, with Incidence Rate Ratios (IRR) of 2.16 (95% CI 1.81-2.58) and 2.37 (95% CI 2.01-2.81) for VL-HIV+ and PKDL cases respectively. A sensitivity analysis showed the strength of the association to be similar in each of the six included subdistricts. CONCLUSIONS: These findings indicate the importance of VL-HIV+ patients as infectious reservoirs for Leishmania, and suggest that they represent a threat equivalent to PKDL patients towards the VL elimination initiative on the Indian subcontinent, therefore warranting a similar focus

A comparative transcriptome analysis of Rhizobium etli bacteroids: specific gene expression during symbiotic nongrowth

Rhizobium etli occurs either in a nitrogen-fixing symbiosis with its host plant, Phaseolus vulgaris, or free-living in the soil. During both conditions, the bacterium has been suggested to reside primarily in a nongrowing state. Using genome-wide transcriptome profiles, we here examine the molecular basis of the physiological adaptations of rhizobia to nongrowth inside and outside of the host. Compared with exponentially growing cells, we found an extensive overlap of downregulated growth-associated genes during both symbiosis and stationary phase, confirming the essentially nongrovving state of nitrogen-fixing bacteroids in determinate nodules that are not terminally differentiated. In contrast, the overlap of upregulated genes was limited. Generally, actively growing cells have hitherto been used as reference to analyze symbiosis-specific expression. However, this prevents the distinction between differential expression arising specifically from adaptation to a symbiotic lifestyle and features associated with nongrowth in general. Using stationary phase as the reference condition, we report a distinct transcriptome profile for bacteroids, containing 203 induced and 354 repressed genes. Certain previously described symbiosis-specific characteristics, such as the down-regulation of amino acid metabolism genes, were no longer observed, indicating that these features are more likely due to the nongrowing state of bacteroids rather than representing bacteroid-specific physiological adaptations

A Comparative Transcriptome Analysis of Rhizobium etli Bacteroids: Specific Gene Expression During Symbiotic Nongrowth

Rhizobium etli occurs either in a nitrogen-fixing symbiosis with its host plant, Phaseolus vulgaris, or free-living in the soil. During both conditions, the bacterium has been suggested to reside primarily in a non-growing state. Using genome-wide transcriptome profiles, we here examine the molecular basis of the physiological adaptations of rhizobia to non-growth inside and outside of the host. Compared to exponentially growing cells, we found an extensive overlap of downregulated growth-associated genes during both symbiosis and stationary phase, confirming the essentially non-growing state of nitrogen-fixing bacteroids in determinate nodules that are not terminally differentiated. In contrast, the overlap of upregulated genes was limited. Generally, actively growing cells have hitherto been used as reference to analyse symbiosis-specific expression. However, this prevents the distinction between differential expression arising specifically from adaptation to a symbiotic lifestyle and features associated with non-growth in general. Using stationary phase as reference condition, we report a distinct transcriptome profile for bacteroids, containing 203 induced and 354 repressed genes. Certain previously described symbiosis-specific characteristics, such as the downregulation of amino acid metabolism genes, were no longer observed, indicating that these features are more likely due to the non-growing state of bacteroids, rather than representing bacteroid-specific physiological adaptations.status: publishe

Diagnosis of Visceral Leishmaniasis in an Elimination Setting: A Validation Study of the Diagnostic Algorithm in India

Visceral leishmaniasis (VL) is on the verge of elimination on the Indian subcontinent. Nonetheless, the currently low VL-incidence setting brings along new challenges, one of which is the validity of the diagnostic algorithm, based on a combination of suggestive clinical symptoms in combination with a positive rK39 Rapid Diagnostic Test (RDT). With this study, we aimed to assess the positive predictive value of the diagnostic algorithm in the current low-endemic setting in India by re-assessing newly diagnosed VL patients with a qPCR analysis on venous blood as the reference test. In addition, we evaluated the specificity of the rK39 RDT by testing non-VL cases with the rK39 RDT. Participants were recruited in Bihar and Uttar Pradesh, India. VL patients diagnosed based on the diagnostic algorithm were recruited through six primary health care centers (PHCs); non-VL cases were identified through a door-to-door survey in currently endemic, previously endemic, and non-endemic clusters, and tested with rK39 RDT, as well as—if positive—with qPCR on peripheral blood. We found that 95% (70/74; 95% CI 87–99%) of incident VL cases diagnosed at the PHC level using the current diagnostic algorithm were confirmed by qPCR. Among 15,422 non-VL cases, 39 were rK39 RDT positive, reflecting a specificity of the test of 99.7% (95% CI 99.7–99.8%). The current diagnostic algorithm combining suggestive clinical features with a positive rK39 RDT still seems valid in the current low-endemic setting in India

Evaluation of qPCR on blood and skin microbiopsies, peripheral blood buffy coat smear, and urine antigen ELISA for diagnosis and test of cure for visceral leishmaniasis in HIV-coinfected patients in India: a prospective cohort study

Introduction: HIV coinfection presents a challenge for diagnosis of visceral leishmaniasis. Invasive splenic or bone marrow aspiration with microscopic visualisation of Leishmania parasites remains the gold standard for diagnosis of VL in HIV patients. Furthermore, a test-of-cure by splenic or bone marrow aspiration is required as VL-HIV patients are at a high risk of treatment failure. However, there remain financial, implementation and safety costs to these invasive techniques which severely limit their use under field conditions. Methods and analysis: We aim to evaluate blood and skin qPCR, peripheral blood buffy coat smear microscopy and urine antigen ELISA as non- or minimally invasive alternatives for diagnosis and post treatment test-of-cure for visceral leishmaniasis in HIV co-infected patients in India, using a sample of 91 parasitologically confirmed symptomatic VL-HIV patients. Ethics and dissemination: Ethical approval for this study has been granted by The Liverpool School of Tropical Medicine, The Institute of Tropical Medicine in Antwerp, the University of Antwerp, and the Rajendra Memorial Research Institute of Medical Science in Patna. Any future publications will be published in open access journals. CTRI Trial registration number: CTRI/2019/03/01790

Post kala azar dermal leishmaniasis and leprosy prevalence and distribution in the Muzaffarpur health and demographic surveillance site.

IntroductionPost-kala-azar dermal leishmaniasis (PKDL) is a skin manifestation that is a late clinical outcome of visceral leishmaniasis (VL). Its presentation is similar to leprosy, and the differential diagnosis is not always easy. In VL endemic rural areas of Bihar, India, both infectious diseases co-exist. This observational study aimed to determine the prevalence and distribution of both conditions in an area that had until recently been highly endemic for VL.MethodsWe conducted a door-to-door survey in an area that belongs to the Health and Demographic Surveillance Site (HDSS) of Muzaffarpur, Bihar, India. Within the HDSS we selected the villages that had reported the highest numbers of VL cases in preceding years. All consenting household members were screened for skin conditions, and minor conditions were treated on the spot. Upon completion of screening activities at the level of a few villages, a dermatology clinic ("skin camp") was conducted to which suspect leprosy and PKDL patients and other patients with skin conditions requiring expert advice were referred. We studied the association between distance from an index case of leprosy and the probability of disease in the neighborhood by fitting a Poisson model.ResultsWe recorded a population of 33,319, out of which 25,686 (77.1%) were clinically screened. Participation in skin camps was excellent. Most common conditions were fungal infections, eczema, and scabies. There were three PKDL patients and 44 active leprosy patients, equivalent to a prevalence rate of leprosy of 17.1 per 10,000. Two out of three PKDL patients had a history of VL. Leprosy patients were widely spread across villages, but within villages, we found strong spatial clustering, with incidence rate ratios of 6.3 (95% C.I. 1.9-21.0) for household members and 3.6 (95% C.I. 1.3-10.2) for neighbors within 25 meters, with those living at more than 100 meters as the reference category.DiscussionEven in this previously highly VL endemic area, PKDL is a rare condition. Nevertheless, even a single case can trigger a new VL outbreak. Leprosy is also a rare disease, but current prevalence is over 17 times the elimination threshold proclaimed by WHO. Both diseases require continued surveillance. Active case finding for leprosy can be recommended among household members and close neighbors of leprosy patients but would not be feasible for entire populations. Periodic skin camps may be a feasible and affordable alternative

Source Tracing of Leishmania donovani in Emerging Foci of Visceral Leishmaniasis, Western Nepal

We sequenced Leishmania donovani genomes in blood samples collected in emerging foci of visceral leishmaniasis in western Nepal. We detected lineages very different from the preelimination main parasite population, including a new lineage and a rare one previously reported in eastern Nepal. Our findings underscore the need for genomic surveillance

The ongoing risk of Leishmania donovani transmission in eastern Nepal : an entomological investigation during the elimination era

Abstract: Background Visceral leishmaniasis (VL), a life-threatening neglected tropical disease, is targeted for elimination from Nepal by the year 2026. The national VL elimination program is still confronted with many challenges including the increasingly widespread distribution of the disease over the country, local resurgence and the questionable efficacy of the key vector control activities. In this study, we assessed the status and risk of Leishmania donovani transmission based on entomological indicators including seasonality, natural Leishmania infection rate and feeding behavior of vector sand flies, Phlebotomus argentipes, in three districts that had received disease control interventions in the past several years in the context of the disease elimination effort. Methods We selected two epidemiologically contrasting settings in each survey district, one village with and one without reported VL cases in recent years. Adult sand flies were collected using CDC light traps and mouth aspirators in each village for 12 consecutive months from July 2017 to June 2018. Leishmania infection was assessed in gravid sand flies targeting the small-subunit ribosomal RNA gene of the parasite (SSU-rRNA) and further sequenced for species identification. A segment (similar to 350 bp) of the vertebrate cytochrome b (cytb) gene was amplified from blood-fed P. argentipes from dwellings shared by both humans and cattle and sequenced to identify the preferred host. Results Vector abundance varied among districts and village types and peaks were observed in June, July and September to November. The estimated Leishmania infection rate in vector sand flies was 2.2% (1.1%-3.7% at 95% credible interval) and 0.6% (0.2%-1.3% at 95% credible interval) in VL and non-VL villages respectively. The common source of blood meal was humans in both VL (52.7%) and non-VL (74.2%) villages followed by cattle. Conclusions Our findings highlight the risk of ongoing L. donovani transmission not only in villages with VL cases but also in villages not reporting the presence of the disease over the past several years within the districts having disease elimination efforts, emphasize the remaining threats of VL re-emergence and inform the national program for critical evaluation of disease elimination strategies in Nepal