Portable smartphone-based molecular test for rapid detection of Leishmania spp.

PURPOSE: Leishmaniasis, caused by the parasite of the genus Leishmania, is a neglected tropical disease which is endemic in more than 60 countries. In South-East Asia, Brazil, and East Africa, it mainly occurs as kala-azar (visceral leishmaniasis, VL), and subsequently as post kala-azar dermal leishmaniasis (PKDL) in a smaller portion of cases. As stated per WHO roadmap, accessibility to accurate diagnostic methods is an essential step to achieve elimination. This study aimed to test the accuracy of a portable minoo device, a small battery-driven, multi-use fluorimeter operating with isothermal technology for molecular diagnosis of VL and PKDL. METHODS: Fluorescence data measured by the device within 20 min are reported back to the mobile application (or app) via Bluetooth and onward via the internet to a backend. This allows anonymous analysis and storage of the test data. The test result is immediately returned to the app displaying it to the user. RESULTS: The limit of detection was 11.2 genome copies (95% CI) as determined by screening a tenfold dilution range of whole Leishmania donovani genomes using isothermal recombinase polymerase amplification (RPA). Pathogens considered for differential diagnosis were tested and no cross-reactivity was observed. For its diagnostic performance, DNA extracted from 170 VL and PKDL cases, comprising peripheral blood samples (VL, n = 96) and skin biopsies (PKDL, n = 74) from India (n = 108) and Bangladesh (n = 62), was screened. Clinical sensitivity and specificity were 88% and 91%, respectively. CONCLUSION: Minoo devices can offer a convenient, cheaper alternative to other molecular diagnostics. Its easy handling makes it ideal for use in low-resource settings to identify parasite burden.

Feasibility of MinION Nanopore Rapid Sequencing in the Detection of Common Diarrhea Pathogens in Fecal Specimen.

The need for fast detection of etiological agents outside the narrow target range of pathogens that may cause an event of an infectious disease epidemic necessitates rapid sequencing technologies to be implemented in routine diagnostic procedures. We tested the performance of a PCR-free rapid nanopore barcoding assay to detect microbial species by analyzing genomic contents extracted from acute diarrheal case specimens. Sequenced reads were processed in an automated analysis module for species identification, whereas pathogenic subspecies detection was aided by a sequence similarity search against a gene-specific database. Evaluation of assay and analysis parameters (e.g., run-time, sequence length, and species hit abundance level) was carried out using a standard bacterial community for assessing detection accuracy. It was observed that longer sequence length (≥500 nucleotides) along with higher species abundance level (≥1%) can be critical for exclusion of false-negative outcomes, while increased sequencing run-time can affect the proportional abundance of true-positive species. Under optimal parameters, the sensitivity of the rapid assay remained 100% for the detection of a target species in a background of nontarget fecal (diarrheal) DNA that weighed up to 64 times the DNA of the target species. The method was applied to acute diarrheal samples. Among these, 62.5% (5/8) were in agreement with target-specific traditional diagnosis methods for the presence/absence of pathogenic agent(s), 12.5% (1/8) were in disagreement, and pathogenic agents that were not targeted by the traditional methods were revealed by sequencing for 25% (2/8) of samples. These observations suggest that further optimization and evaluation of the rapid nanopore sequencing method could potentiate the widening of the range of pathogens that can be detected in acute diarrheal samples in the context of regular diagnostic needs as well as epidemics.

Rapid Detection of SARS-CoV-2 by Low Volume Real-Time Single Tube Reverse Transcription Recombinase Polymerase Amplification Using an Exo Probe with an Internally Linked Quencher (Exo-IQ).

BACKGROUND: The current outbreak of SARS-CoV-2 has spread to almost every country with more than 5 million confirmed cases and over 300,000 deaths as of May 26, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance. METHODS: We used computational and manual designs to generate a suitable set of reverse transcription recombinase polymerase amplification (RT-RPA) primer and exonuclease probe, internally quenched (exo-IQ), sequences targeting the SARS-CoV-2 N gene. RT-RPA sensitivity was determined by amplification of in vitro transcribed RNA standards. Assay selectivity was demonstrated with a selectivity panel of 32 nucleic acid samples derived from common respiratory viruses. To validate the assay against full-length SARS-CoV-2 RNA, total viral RNA derived from cell culture supernatant and 19 nasopharyngeal swab samples (8 positive and 11 negative for SARS-CoV-2) were screened. All results were compared to established RT-qPCR assays. RESULTS: The 95% detection probability of the RT-RPA assay was determined to be 7.74 (95% CI: 2.87-27.39) RNA copies per reaction. The assay showed no cross-reactivity to any other screened coronaviruses or respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n = 20). CONCLUSIONS: With a run time of 15 to 20 minutes and first results being available in under 7 minutes for high RNA concentrations, the reported assay constitutes one of the fastest nucleic acid based detection methods for SARS-CoV-2 to date and may provide a simple-to-use alternative to RT-qPCR for first-line screening at the point of need.

Serological Analysis of Herpes B Virus at Individual Epitope Resolution: From Two-Dimensional Peptide Arrays to Multiplex Bead Flow Assays.

Macacine herpesvirus or B Virus (BV) is a zoonotic agent that leads to high mortality rates in humans if transmitted and untreated. Here, BV is used as a test case to establish a two-step procedure for developing high throughput serological assays based on synthetic peptides. In step 1, peptide microarray analysis of 42 monkey sera (30 of them tested BV positive by ELISA) revealed 1148 responses against 369 different peptides. The latter could be grouped into 142 different antibody target regions (ATRs) in six different glycoproteins (gB, gC, gD, gG, gH, and gL) of BV. The high number of newly detected ATRs was made possible inter alia by a new preanalytical protocol that reduced unspecific binding of serum components to the cellulose-based matrix of the microarray. In step 2, soluble peptides corresponding to eight ATRs of particularly high antigenicity were synthesized and coupled to fluorescently labeled beads, which were subsequently employed in immunochemical bead flow assays. Their outcome mirrored the ELISA results used as reference. Hence, convenient, fast, and economical screening of arbitrarily large macaque colonies for BV infection is now possible. The study demonstrates that a technology platform switch from two-dimensional high-resolution peptide arrays used for epitope discovery to a readily available bead array platform for serology applications is feasible.

Development of Mobile Laboratory for Viral Hemorrhagic Fever Detection in Africa.

Background: A mobile laboratory transportable on commercial flights was developed to enable local response to viral hemorrhagic fever outbreaks. Methods: The development progressed from use of mobile real-time reverse-transcription polymerase chain reaction to mobile real-time recombinase polymerase amplification. In this study, we describe various stages of the mobile laboratory development. Results: A brief overview of mobile laboratory deployments, which culminated in the first on-site detection of Ebola virus disease (EVD) in March 2014, and their successful use in a campaign to roll back EVD cases in Conakry in the West Africa Ebola virus outbreak are described. Conclusions: The developed mobile laboratory successfully enabled local teams to perform rapid disgnostic testing for viral hemorrhagic fever.

Development of a pan-rickettsial molecular diagnostic test based on recombinase polymerase amplification assay.

Rickettsioses are zoonotic vector-transmitted bacterial infections leading to flu-like symptoms and can progress to severe illness in humans. The gold standard for diagnosis of rickettsial infections is the indirect immunofluorescence assay, a serological method which is not suitable for pathogen identification during the acute phase of the disease. Therefore, several real-time PCR assays were developed. These assays are very sensitive, but require high-equipped laboratories and well-trained personnel. Hence, in this study, a rapid point-of-need detection method was developed to detect all Rickettsia species. The 23S and 16S rRNA genes were targeted to develop a recombinase polymerase amplification (RPA) assay. Both 23S and 16S_RPA assays required between seven to ten minutes to amplify and detect one or ten DNA molecules/reaction, respectively. The 16S_RPA assay detected all tested species, whereas the 23S_RPA assay identified only species of the spotted fever and transitional rickettsial groups. All results were compared with real-time PCR assays directed against the same rickettsial genes. The RPA assays are easy to handle and produced quicker results in comparison to real-time PCRs. Both RPA assays were implemented in a mobile suitcase laboratory to ease the use in rural areas. This method can help to provide rapid management of rickettsial infections.

Recombinase polymerase amplification assay for rapid detection of lumpy skin disease virus.

BACKGROUND: Lumpy skin disease virus (LSDV) is a Capripoxvirus infecting cattle and Buffalos. Lumpy skin disease (LSD) leads to significant economic losses due to hide damage, reduction of milk production, mastitis, infertility and mortalities (10 %). Early detection of the virus is crucial to start appropriate outbreak control measures. Veterinarians rely on the presence of the characteristic clinical signs of LSD. Laboratory diagnostics including virus isolation, sequencing and real-time polymerase chain reaction (PCR) are performed at well-equipped laboratories. In this study, a portable, simple, and rapid recombinase polymerase amplification (RPA) assay for the detection of LSDV-genome for the use on farms was developed. RESULTS: The LSDV RPA assay was performed at 42 °C and detected down to 179 DNA copies/reaction in a maximum of 15 min. Unspecific amplification was observed with neither LSDV-negative samples (n = 12) nor nucleic acid preparations from orf virus, bovine papular stomatitis virus, cowpoxvirus, Peste des petits ruminants and Blue tongue virus (serotypes 1, 6 and 8). The clinical sensitivity of the LSDV RPA assay matched 100 % (n = 22) to real-time PCR results. In addition, the LSDV RPA assay detected sheep and goat poxviruses. CONCLUSION: The LSDV RPA assay is a rapid and sensitive test that could be implemented in field or at quarantine stations for the identification of LSDV infected case.

Detection of Zika virus in Brazilian patients during the first five days of infection - urine versus plasma.

Advantages of testing for Zika virus (ZIKV) in urine have been reported, such as the persistence of ZIKV in this type of specimen for up to 20 days after ZIKV disease onset. We investigate 61 patients in the first 5 days post-symptom onset and find more patients testing positive for ZIKV in plasma samples (n=46), than in corresponding urine samples (n=37). For patients respectively testing positive in both plasma and urine (n=28), respective viral loads appeared similar.

Fast and on-site animal species identification in processed meat via centrifugal microfluidics and isothermal amplification.

We present a novel centrifugal microfluidic approach to rapidly identify animal species in meat products. The workflow requires a centrifugal cartridge for DNA extraction and for preparation of a recombinant polymerase amplification (RPA) reaction, a programmable centrifuge for processing the cartridge and an isothermal reader to perform the RPA. Liquid reagents are pre-stored on the cartridge and the meat sample can be added directly without any pre-treatment. With this system, we are able to identify six different animal species in a single run within one hour. In pork salami containing horse, turkey, sheep, chicken and beef meat, it was possible to identify species levels as low as 0.01%. In beef salami and cooked pork sausages 0.1% of foreign meat could be detected. This novel workflow enables rapid and sensitive species identification in processed meat at the point of need.

Detection of pathogenic Leptospira with rapid extraction followed by recombinase polymerase amplification (RPA) and quantitative polymerase chain reaction (qPCR) assay-A comprehensive study from Sri Lanka.

Leptospirosis is the most widespread zoonosis in the world. The disease is more prevalent in tropical regions where the majority of developing countries are located. Leptospirosis is considered a protean manifestation zoonosis with severity of the disease ranging from a mild febrile illness to a severe and life-threatening illness. Clinical symptoms of leptospirosis overlap with other tropical febrile illnesses. Early, rapid, and definitive diagnosis is important for effective patient management. Since Polymerase Chain Reaction (PCR)-based assays are not readily available in most clinical settings, there is a need for an affordable, simple, and rapid diagnostic test. Quantitative PCR (qPCR) and Recombinase Polymerase Amplification (RPA) were implemented at the Faculty of Medicine, University of Kelaniya, and a prospective study to evaluate RPA for diagnosis of acute phase of leptospirosis was conducted. Results indicate that RPA and qPCR were positive in 81% (98/121) of the total positive and acute clinical samples. Of the 81 positive MAT confirmed patients 60 (74%) and 53 (65%) were positive with qPCR and RPA respectively. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity-70% and specificity-87%) of RPA compared to MAT as the reference gold standard. Results further suggest that there is no significant difference between the two assays, qPCR and RPA-SwiftX (P = 0.40). Laboratory procedures for the extraction and detection by qPCR in the laboratory have been optimized to obtain results within 6 hours. However, the RPA-SwiftX method under field conditions took 35 minutes. The RPA-SwiftX method could replace the qPCR which shows similar sensitivity and specificity. Therefore, RPA established under the current study presents a powerful tool for the early and rapid diagnosis of leptospirosis at point-of-care.

Validation of the efficacy of air purifiers using molecular techniques.

The importance of air purifiers has increased in recent years, especially with the "coronavirus disease 2019" pandemic. The efficacy of air purifiers is usually determined under laboratory conditions before widespread application. The standard procedure for testing depends on virus cultivation and titration on cell culture. This, however, requires several days to deliver results. The aim of this study was to establish a rapid molecular assay which can differentiate between intact infectious and distorted non-infectious virus particles. Feline Coronavirus was selected as model for screening. First the samples were pretreated with enzymes (universal nuclease and RNase cocktail enzyme mixture) or viability dye (propidium monoazide) to eliminate any free nucleic acids. The ribonucleic acid (RNA) from intact virus was released via magnetic beads-based extraction, then the amount of the RNA was determined using real-time reverse transcription polymerase chain reaction (RT-PCR) or reverse transcription recombinase-aided amplification (RT-RAA). All results were compared to the infectivity assay based on the calculation of the 50% tissue culture infectious dose (TCID50). The nuclease has eliminated 100% of the free Feline Coronavirus RNA, while propidium monoazide underperformed (2.3-fold decrease in free RNA). Both RT-RAA and real-time RT-PCR produced similar results to the infectivity assay on cell culture with limit of detection of 102 TCID50/mL. Two UV-C air purifiers with prosperities of 100% inactivation of the viruses were used to validate the established procedure. Both real-time RT-PCR and RT-RAA were able to differentiate between intact virus particles and free RNA. To conclude, this study revealed a promising rapid method to validate the efficacy of air purifiers by combining enzymatic pretreatment and molecular assays.

Rapid Reverse Purification DNA Extraction Approaches to Identify Microbial Pathogens in Wastewater.

Wastewater monitoring became a promising solution in the early detection of outbreaks. Despite the achievements in the identification of pathogens in wastewater using real-time PCR, there is still a lack of reliable rapid nucleic acid extraction protocols. Therefore, in this study, samples were subjected to alkali, proteinase K and/or bead-beating followed by reverse purification magnetic beads-based separation. Wastewater samples spiked with S. aureus, E. coli and C. parvum were used as examples for Gram-positive and -negative bacteria and protozoa, respectively. All results were compared with a spin column technology as a reference method. Proteinase K with bead beating (vortexing with 0.1 mm glass beads for three minutes) was particularly successful for bacterial DNA extraction (three- to five-fold increase). The most useful extraction protocol for protozoa was pre-treatment with proteinase K (eight-fold increase). The selected methods were sensitive as far as detecting one bacterial cell per reaction for S. aureus, ten bacterial cells for E. coli and two oocysts for C. parvum. The extraction reagents are cold chain independent and no centrifuge or other large laboratory equipment is required to perform DNA extraction. A controlled validation trial is needed to test the effectiveness at field levels.

Revisiting the diagnosis and treatment of Para Kala-azar Dermal Leishmaniasis in the endemic foci of Bangladesh.

Para Kala-azar Dermal Leishmaniasis (Para-KDL) manifests the concomitant presence of Post Kala-azar Dermal Leishmaniasis and Visceral Leishmaniasis and works as a reservoir of infection. The study discusses the cases and their management and aims to address the gaps within existing methods of diagnosis and treatment. This retrospective cross-sectional study discusses 16 Para-KDL cases with one-year follow-up data, treated between 2012-2021 at the Surya Kanta Kala-azar Research Center, Bangladesh. We collected data from hospital records and used STATA 16 to analyze and see the frequency distribution and variable means. We found five patients without any history of kala-azar infection. All the patients were treated with 20 mg/kg Liposomal Amphotericin B in 4 divided doses except one with a history of AmBisome hypersensitivity. One year after treatment, all patients were free from skin lesions, with no hepatosplenomegaly, and observed significant improvement in BMI and hemoglobin levels. The Para-KDL patients are challenging to diagnose, and the relapse and treatment failure leishmania patients might have belonged to this rare group, contributing to their poor prognosis. Therefore, developing an appropriate diagnostic workflow and a new drug regimen is essential to sustain the success of our elimination efforts.

Assessment of pan-Leishmania detection by recombinase polymerase amplification assay.

The spread of vector habitats along with increasing human mobility can introduce atypical Leishmania species and hence can challenge existing diagnostic practices for rapid detection of active infection with species outside the narrow target range. Here we assessed the pan-Leishmania detection ability of isothermal recombinase polymerase amplification (RPA) assays targeting 18S rRNA gene, cathepsin L-like cysteine proteinase B (Cpb) gene, and kinetoplast minicircle DNA (kDNA) regions. While the lowest limit of detection of the 18S rRNA-RPA and Cpb-RPA assays were estimated as 12 and 17 standard DNA molecules, respectively, both assays could amplify genomic DNA of 7 pathogenic Leishmania species. Evaluation of 18S rRNA-RPA and our previously developed kDNA-RPA assays on 70 real-time PCR-positive leishmaniasis samples of varying pathologies resulted in sensitivity rates of 35.71% and 88.57%, respectively, while the combined sensitivity was 98.57%. Combinatorial application of 18S rRNA-RPA and kDNA-RPA assays can be recommended for further diagnostic assessments.

Use of Envelope Domain III Protein for the Detection of IgG Type Antibodies Specific to Zika Virus by Indirect ELISA.

Zika virus (ZIKV) diagnostics are crucial for proper antenatal and postnatal care and also for surveillance and serosurvey studies. Since the viremia during ZIKV infection is fleeting, serological testing is highly valuable to inform diagnosis. However, current serology tests using whole virus antigens frequently suffer from cross reactivity issues, delays, and technical complexity, especially in low and middle income countries (LMICs) and endemic countries. Here, we describe an indirect ELISA to detect specific IgG antibodies using the ZIKV envelope domain III (EDIII) protein expressed in Drosophila S2 cells as an immunogen. Using a total of 367 clinical samples, we showed that the EDIII-ELISA was able to detect IgG antibodies against ZIKV with high sensitivity of 100.0% and specificity of 94.7% when compared to plaque reduction neutralization tests (PRNTs) as the gold standard and using 0.208 as the cut-off OD value. These results show the usefulness of the recombinant envelope domain III as an alternative to standard whole virus proteins for ZIKV diagnostics as it improves the sensitivity and specificity of IgG ELISA assay when used as an immunogen. This method should, therefore, be extended to serological diagnostic techniques for other members of the flavivirus genus and for use in IgM diagnostic testing.

Decision-support tools to build climate resilience against emerging infectious diseases in Europe and beyond.

Climate change is one of several drivers of recurrent outbreaks and geographical range expansion of infectious diseases in Europe. We propose a framework for the co-production of policy-relevant indicators and decision-support tools that track past, present, and future climate-induced disease risks across hazard, exposure, and vulnerability domains at the animal, human, and environmental interface. This entails the co-development of early warning and response systems and tools to assess the costs and benefits of climate change adaptation and mitigation measures across sectors, to increase health system resilience at regional and local levels and reveal novel policy entry points and opportunities. Our approach involves multi-level engagement, innovative methodologies, and novel data streams. We take advantage of intelligence generated locally and empirically to quantify effects in areas experiencing rapid urban transformation and heterogeneous climate-induced disease threats. Our goal is to reduce the knowledge-to-action gap by developing an integrated One Health-Climate Risk framework.

Evaluation of a simple ultrafiltration method for concentration of infective canine parvovirus and feline coronavirus from cell culture supernatants.

Enrichment of viral infectious titers following its propagation by cell culture is desirable for various experimental studies. The performance of an ultrafiltration (UF) process to concentrate infectious titers of non-enveloped Canine parvovirus 2 (CPV-2) and enveloped Feline coronavirus (FCoV) obtained from cell culture supernatants was evaluated in this study, and compared with ultracentrifugation (UC) process. A mean gain of > 1.0 log10 TCID50/mL was obtained for CPV-2 with UF, which was comparable with the gain obtained by UC. On the other hand, the gain was lower (0.7-1.0 log10 TCID50/mL) for FCoV with UF in contrast to UC (> 2.0 log10 TCID50/mL). However, the lower retentate volume following UC (∼120 fold) compared to that following UF (∼10 fold) for either of the viruses suggests a trend of increased infectious titer retention in UF concentrates relative to UC concentrates. The simplistic UF process evaluated here thus has the potential for use in applications requiring increased infectious titers of CPV-2 and FCoV.

Efficacy of Liming Forest Soil in the Context of African Swine Fever Virus.

Since September 2020, Germany has experienced the first ever outbreak of African swine fever (ASF). The first known cases occurred exclusively in wild boar in forest areas in Brandenburg and Saxony; in July 2021, infected domestic pigs were also confirmed for the first time. As wild boar are considered the main reservoir for the virus in the European region, an effective interruption of this infection chain is essential. In particular, the removal and safe disposal of infected carcasses and the direct disinfection of contaminated, unpaved ground are priorities in this regard. For the disinfection, highly potent as well as environmentally compatible disinfectants must be used, which are neither influenced in their effectiveness by the soil condition nor by increased organic contamination. Thus, in this study, slaked lime, milk of lime and quicklime (1% to 10% solutions) were selected for efficacy testing against the test virus recommended by the German Veterinary Society (DVG), Modified Vaccinia Ankara virus (MVAV), and ASF virus (ASFV) in conjunction with six different forest soils from Saxony in two different soil layers (top soil and mineral soil) each. In summary, 10% of any tested lime type is able to inactivate both MVAV and ASFV under conditions of high organic load and independent of the water content of the soil. At least a 4 log reduction of the virus titer in all tested forest soil types and layers and by all applied lime types was observed. In conclusion, the high efficacy and suitability of all tested lime products against both viruses and in the presence of high organic load in forest soil can be confirmed and will help to control ASF spread.

Assessment of Risk of Exposure to Leishmania Parasites among Renal Disease Patients from a Renal Unit in a Sri Lankan Endemic Leishmaniasis Focus.

Leishmania donovani causes both cutaneous and visceral leishmaniasis (CL and VL) in Sri Lanka, where chronic kidney disease (CKD) and kidney transplant recipients' (KTR) geographical areas overlap. This study aimed to determine the risk of exposure to Leishmania infection among renal patients. This cross-sectional study in a renal unit assessed clinical symptoms and signs of CL and VL in recipients of blood/kidney or immunosuppressives. Sera were tested with Leishmania-specific DAT and rK-39 ELISA. There were 170 participants. A total of 84.1% (n = 143) were males (CKD: 101, KTR; 42, mean age 45) and 27 were females (females: CKD: 23, KTR: 4, mean age 39 years). Recipients of blood transfusion/s within last 2 years: 75.9% (CKD: 115, KTR: 14), on immunosuppressive therapy: 34.1% (CKD: 13, KTR: 45). Two CKD patients repeatedly showed clear positive titres (1: 12,800 and 1: 3200) with Leishmania-DAT and another two (CKD) became marginally positive with rK39-ELISA. Prevalence of anti-Leishmania antibodies: 2.4% (4/170). All four patients were clinically asymptomatic and were recipients of recent blood transfusions. Attributable risk of exposure to Leishmania infection through blood transfusions was 0.032, OR 2.99 (95% CI = 0.16 to 56.45, p = 0.47). Therefore, routine screening of kidney/blood donors and CKD and KTR patients in Sri Lanka may not be necessary.

First Report on Ovine Paratuberculosis in the Sudan: Diagnosis Using Different Techniques.

Paratuberculosis (PTB) has been reported in the Sudan in cattle and goats for more than 50 years but has never been reported in sheep. However, suspicion of the disease in a breeding flock of sheep in Khartoum North locality was made due to a history of unknown cause of loss of weight. Blood and faecal samples were collected from all animals (N = 59): harvested sera were tested for anti-Mycobacterium avium subsp. paratuberculosis (MAP) antibodies by Enzyme Linked Immunosorbent Assay (ELISA); faeces were screened for acid-fast bacilli by Ziehl-Neelsen staining, tested for MAP DNA by recombinase polymerase amplification (RPA) and some faecal samples were cultured for MAP isolation. Typical MAP acid-fast bacilli were seen in 10.2% (6/59) of the faecal smears, 37.5% of the tested faecal samples (12/32) were positive for MAP DNA and only 3 (5.1%) animals were seropositive for MAP. MAP positive cultures were obtained from 2 out the 6 samples showing typical MAP acid-fast bacilli; the isolates were confirmed by real-time PCR and sequencing. As sheep are animals of utmost economic importance as the main export animals for the country, this first report of ovine PTB warrants special considerations and more investigations for planning control programmes of the disease.