Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible ...and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19-100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16-94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55-89.16%) for VL samples, 100% (95%CI, 69.15-100%) for RVL samples, and 52.5% (95% CI, 36.13-68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98-100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L. donovani parasites in VL, PKDL and RVL patients, as well as being capable of assessing response to treatment. As such, this real time PCR assay represents an important contribution in efforts to eliminate VL.
Stunting, a severe and multigenerational growth impairment, globally affects 22% of children under the age of 5 years. Stunted children have altered gut bacterial communities with higher proportions ...of Proteobacteria, a phylum with several known human pathogens. Despite the links between an altered gut microbiota and stunting, the role of bacteriophages, highly abundant bacterial viruses, is unknown. Here, we describe the gut bacterial and bacteriophage communities of Bangladeshi stunted children younger than 38 months. We show that these children harbor distinct gut bacteriophages relative to their non-stunted counterparts. In vitro, these gut bacteriophages are infectious and can regulate bacterial abundance and composition in an age-specific manner, highlighting their possible role in the pathophysiology of child stunting. Specifically, Proteobacteria from non-stunted children increased in the presence of phages from younger stunted children, suggesting that phages could contribute to the bacterial community changes observed in child stunting.
Chikungunya fever is a mosquito‐borne disease cause of persistent arthralgia. The current diagnosis of Chikungunya virus (CHIKV) relies on a conventional reverse transcription polymerase chain ...reaction assay. Reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) is a rapid and simple tool used for DNA‐based diagnosis of a variety of infectious diseases. In this study, we established an RT‐LAMP system to recognize CHIKV by targeting the envelope protein 1 (E1) gene that could also detect CHIKV at a concentration of 8 PFU without incorrectly detecting other mosquito‐borne viruses. The system also amplified the E1 genome in the serum of CHIKV‐infected mice with high sensitivity and specificity. Moreover, we established a dry RT‐LAMP system that can be transported without a cold chain, which detected the virus genome in CHIKV‐infected patient samples with high accuracy. Thus, the dry RT‐LAMP system has great potential to be applied as a novel CHIKV screening kit in endemic areas.
A portable, simple and cost‐effective dry RT‐LAMP system specific for the E1 region of CHIKV. Highly sensitive and specific to CHIKV, but not to other mosquito‐borne viruses. A rapid screening platform suitable for on‐site diagnosis to detect CHIKV in human clinical samples.
Leishmania donovani (LD) is a protozoan parasite transmitted to humans from sand flies, which causes Visceral Leishmaniasis (VL). Currently, the diagnosis is based on presence of the anti-LD ...antibodies and clinical symptoms. Molecular diagnosis would require real-time PCR, which is not easy to implement at field settings. In this study, we report on the development and testing of a recombinase polymerase amplification (RPA) assay for the detection of LD.
A genomic DNA sample was applied to determine the assay analytical sensitivity. The cross-reactivity of the assay was tested by DNA of Leishmania spp. and of pathogens considered for differential diagnosis. The clinical performance of the assay was evaluated on LD positive and negative samples. All results were compared with real-time PCR. To allow the use of the assay at field settings, a mobile suitcase laboratory (56 × 45.5 × 26.5 cm) was developed and operated at the local hospital in Mymensingh, Bangladesh.
The LD RPA assay detected equivalent to one LD genomic DNA. The assay was performed at constant temperature (42 °C) in 15 min. The RPA assay also detected other Leishmania species (L. major, L. aethiopica and L. infantum), but did not identify nucleic acid of other pathogens. Forty-eight samples from VL, asymptomatic and post-kala-azar dermal leishmaniasis subjects were detected positive and 48 LD-negative samples were negative by both LD RPA and real-time PCR assays, which indicates 100 % agreement. The suitcase laboratory was successfully operated at the local hospital by using a solar-powered battery. DNA extraction was performed by a novel magnetic bead based method (SpeedXtract), in which a simple fast lysis protocol was applied. Moreover, All reagents were cold-chain independent.
The mobile suitcase laboratory using RPA is ideal for rapid sensitive and specific detection of LD especially at low resource settings and could contribute to VL control and elimination programmes.
Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in animals with zoonotic potential; it has been linked to many chronic diseases in humans, especially gastrointestinal ...diseases (GID). MAP has been extensively studied in Europe and America, but little reports were published from Africa. Sudan is a unique country with close contact between humans and livestock. Despite such interaction, the one health concept is neglected in dealing with cases of humans with GID. In this study, patients admitted to the reference GID hospital in the Sudan over a period of 8 months were screened for presence of MAP in their faeces or colonic biopsies. A total of 86 patients were recruited for this study, but only 67 were screened for MAP, as 19 did not provide the necessary samples for analysis. Both real-time PCR and culture were used to detect MAP in the collected samples and the microbial diversity in patients´ faecal samples was investigated using 16S rDNA nanopore sequencing. In total, 27 (40.3%) patients were MAP positive: they were 15 males and 12 females, of ages between 21 and 80 years. Logistic regression analysis revealed no statistical significance for all tested variables in MAP positive patients (occupation, gender, contact with animal, milk consumption, chronic disease, etc.). A unique microbiome profile of MAP-positive patients in comparison to MAP-negative was found. These findings suggest that a considerable proportion of the population could be MAP infected or carriers. Therefore, increase awareness at community level is urgently needed to decrease the risk of MAP at human/animal interface. This study represents the first report of MAP in humans in the Sudan; nevertheless, a better view of the situation of MAP in humans in the country requires a larger study including patients with other conditions.
A rapid, cost-effective, and simple nucleic acid isolation technique coupled with a point-of-need DNA amplification assay is a desirable goal for programmatic use. For diagnosis of Visceral ...Leishmaniasis (VL), Recombinase Polymerase Amplification (RPA) rapid tests for the detection of Leishmania DNA are versatile and have operational advantages over qPCR. To facilitate the delivery of the RPA test at point-of-need for VL diagnosis, we compared two rapid DNA extraction methods, SwiftDx (SX) and an in-house Boil and Spin (BS) method, coupled with RPA amplification, versus more widely used methods for DNA extraction and amplification, namely Qiagen (Q) kits and qPCR, respectively. A total of 50 confirmed VL patients and 50 controls, matched for age and gender, were recruited from Mymensingh, Bangladesh, a region highly endemic for VL. Blood samples were collected from each participant and DNA was extracted using Q, SX and BS methods. Following DNA extraction, qPCR and RPA assays were performed to detect
in downstream analysis. No significant differences in sensitivity of the RPA assay were observed between DNA extraction methods, 94.00% (95% CI: 83.45-98.75%), 90% (95% CI: 78.19-96.67%), and 88% (95% CI: 75.69-95.47%) when using Q, SX, and BS, respectively. Similarly, using qPCR, no significant differences in sensitivity were obtained when using Q or SX for DNA extraction, 94.00% (95% CI: 83.45-98.75%) and 92.00% (80.77-97.78%), respectively. It is encouraging that RPA and qPCR showed excellent agreement (k: 0.919-0.980) when different extraction methods were used and that the DNA impurities using BS had no inhibitory effect on the RPA assay. Furthermore, significantly higher DNA yields were obtained using SX and BS versus Q; however, a significantly higher parasite load was detected using qPCR when DNA was extracted using Q versus SX. Considering the cost, execution time, feasibility, and performance of RPA assay, rapid extraction methods such as the Boil and Spin technique appear to have the potential for implementation in resource-limited endemic settings. Further clinical research is warranted prior to broader application.
With reduced prevalence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC), direct and field deployable diagnostic tests are needed to implement an effective diagnostic and surveillance ...algorithm for post-elimination VL control. In this regard, here we investigated the diagnostic efficacies of a loop-mediated isothermal amplification (LAMP) assay (Loopamp™
Detection Kit, Eiken Chemical CO., Ltd, Japan), a real-time quantitative PCR assay (qPCR) and the
antigen ELISA (CLIN-TECH, UK) with different sampling techniques and evaluated their prospect to incorporate into post-elimination VL control strategies. Eighty clinically and rK39 rapid diagnostic test confirmed VL cases and 80 endemic healthy controls were enrolled in the study. Peripheral blood and dried blood spots (DBS) were collected from all the participants at the time of diagnosis. DNA was extracted from whole blood (WB) and DBS
silica columns (QIAGEN) and boil & spin (B&S) methods and tested with qPCR and Loopamp. Urine was collected from all participants at the time of diagnosis and was directly subjected to the
antigen ELISA. 41 patients were followed up and urine samples were collected at day 30 and day 180 after treatment and ELISA was performed. The sensitivities of the Loopamp-WB(B&S) and Loopamp-WB(QIA) were 96.2% (95% CI 89·43-99·22) and 95% (95% CI 87·69-98·62) respectively. The sensitivity of Loopamp-DBS(QIA) was 85% (95% CI 75·26- 92·00). The sensitivities of the qPCR-WB(QIA) and qPCR-DBS(QIA) were 93.8% (95% CI 86·01-97·94) and 72.5% (95% CI 61·38-81·90) respectively. The specificity of all molecular assays was 100%. The sensitivity and specificity of the
antigen ELISA were 97.5% (95% CI 91·47-99·70) and 91.95% (95% CI 84·12-96·70) respectively. The
antigen ELISA depicted clinical cure at day 180 in all the followed-up cases. Efficacy and sustainability identify the Loopamp-WB(B&S) and the
antigen ELISA as promising and minimally invasive VL diagnostic tools to support VL diagnostic and surveillance activities respectively in the post-elimination era.
Visceral leishmaniasis (VL) caused by dimorphic Leishmania species is a parasitic disease with high socioeconomic burden in endemic areas worldwide. Sustaining control of VL in terms of proper and ...prevailing immunity development is a global necessity amid unavailability of a prophylactic vaccine. Screening of experimental proteome of the human disease propagating form of Leishmania donovani (amastigote) can be more pragmatic for in silico mining of novel vaccine candidates.
By using an immunoinformatic approach, CD4+ and CD8+ T cell-specific epitopes from experimentally reported L. donovani proteins having secretory potential and increased abundance in amastigotes were screened. A chimera linked with a Toll-like receptor 4 (TLR4) peptide adjuvant was constructed and evaluated for physicochemical characteristics, binding interaction with TLR4 in simulated physiological condition and the trend of immune response following hypothetical immunization.
Selected epitopes from physiologically important L. donovani proteins were found mostly conserved in L. infantum, covering theoretically more than 98% of the global population. The multi-epitope chimeric vaccine was predicted as stable, antigenic and non-allergenic. Structural analysis of vaccine-TLR4 receptor docked complex and its molecular dynamics simulation suggest sufficiently stable binding interface along with prospect of non-canonical receptor activation. Simulation dynamics of immune response following hypothetical immunization indicate active and memory B as well as CD4+ T cell generation potential, and likely chance of a more Th1 polarized response.
The methodological approach and results from this study could facilitate more informed screening and selection of candidate antigenic proteins for entry into vaccine production pipeline in future to control human VL.
Recombinant fusion proteins are now commonly used to detect circulating antibodies for the serodiagnosis of visceral leishmaniasis (VL) in Asia, Africa and the Americas. Although simple, these tests ...still require blood collection and their use in remote settings can be limited due to the need of collection devices, serum fractionation instrument and generation of biohazardous waste. The development of an accurate and non-invasive diagnostic algorithm for VL, such as could be achieved with urine, is desirable.
We enrolled 87 VL patients and 81 non-VL individuals, including 33 healthy endemic controls, 16 healthy non-endemic controls, 16 disease controls and 16 tuberculosis (TB) patients. We compared the efficacy of recombinant antigens rK28, rK39 and rKRP42 for the diagnosis of VL when either serum or urine were used to develop antibody-detection ELISA.
As expected, each of the antigens readily detected antibodies in the serum of VL patients. rK28 ELISA showed the highest sensitivity (98.9 %), followed by rK39 and rKRP42 ELISA (97.7 and 94.4 %, respectively); overall specificity was > 96 %. When urine was used as the test analyte, only a marginal drop in sensitivity was observed, with rK28 ELISA again demonstrating the greatest sensitivity (95.4 %), followed by rK39 and rKRP42 ELISA, respectively. Again, the overall specificity was > 96 %.
Our data indicate the potential for using urine in the diagnosis of VL. Detection of antibodies against rK28 demonstrated the greatest sensitivity. Together, our results indicate that rK28-based antibody detection tests using urine could provide a completely non-invasive tool amenable for diagnosis of VL in remote locations.
Re-vaccinations against canine distemper virus (CDV) are commonly performed in 3-year intervals. The study's aims were to determine anti-CDV antibodies in healthy adult dogs within 28 days of ...vaccination against CDV, and to evaluate factors associated with the presence of pre-vaccination antibodies and with the antibody response to vaccination. Ninety-seven dogs, not vaccinated within 1 year before enrollment, were vaccinated with a modified live CDV vaccine. A measurement of the antibodies was performed before vaccination (day 0), on day 7, and 28 after the vaccination by virus neutralization. A response to vaccination was defined as a ≥4-fold titer increase by day 28. Fisher's exact test was used to determine factors associated with a lack of antibodies and vaccination response. In total, 94.8% of the dogs (92/97; CI 95%: 88.2-98.1) had antibodies (≥10) prior to vaccination. A response to vaccination was not observed in any dog. Five dogs were considered humoral non-responders; these dogs neither had detectable antibodies before, nor developed antibodies after vaccination. Young age (<2 years) was significantly associated with a lack of pre-vaccination antibodies (
= 0.018; OR: 26.825; 95% CI: 1.216-1763.417). In conclusion, necessity of re-vaccination in adult healthy dogs should be debated and regular vaccinations should be replaced by antibody detection.
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