Resource-limited Laboratories Research Articles

Update on molecular diagnosis of human leptospirosis

Abstract Background Leptospirosis, caused by pathogenic Leptospira spp., is a widespread zoonotic disease worldwide. Early diagnosis is required for proper patient management and reducing leptospirosis morbidity and mortality. Objective To summarize current literature regarding commonly used and new promising molecular approaches to Leptospira detection and diagnostic tests of human leptospirosis. Method The relevant articles in Leptospira and leptospirosis were retrieved from MEDLINE (PubMed) and Scopus. Results Several molecular techniques have been developed for diagnosis of human leptospirosis. Polymerase chain reaction-based techniques targeting on either lipL32 or 16S rRNA (rrs) gene are most commonly used to detect leptospiral DNA in various clinical specimens. Whole blood and urine are recommended specimens for suspected cases in the first (acute) and the second (immune) phases, respectively. Isothermal amplification with less expensive instrument is an alternative DNA detection technique that may be suitable for resource-limited laboratories. Conclusion Detection of leptospiral DNA in clinical specimens using molecular techniques enhances sensitivity for diagnosis of leptospirosis. The efficient and robust molecular detection especially in the early leptospiremic phase may prompt early and appropriate treatment leading to reduced morbidity and mortality of patients with leptospirosis.

Progress In Knowledge Of Microscopic Tuberculosis Diagnosis Laboratory Training Participants

Indonesia is the second highest tuberculosis (TB)-burden country in the world. TB affects men and women in productive ages. Early and accurate diagnosis of TB is crucial in TB management because it allows prompt treatment with appropriate regimen. Sputum smear microscopy is the most common method used in resource-limited laboratories. The aim of this study is to assess whether adult learning method with student-centered approach can improve knowledge in laboratory techniques in microscopic TB diagnosis. It was a quasi experimental research with one group pretest and posttest design. Pretest was conducted before the training started. During the training, participants attended lectures, discussion sessions, and hands-on laboratory activities. After training, participants were asked to respond to posttest. There were 33 participants of this training (n=33). Paired t-test analysis showed significant difference between pretest and posttest scores (p=6.5x10-9). Variance between pretest scores was 4.00 and variance between posttest scores were 1.34. Pearson correlation coefficient was 0.45. Regardless their level of knowledge before training, participants could achieve relatively equivalent results after training. Significant difference between pre- and post-training test scores indicated that the learning method was effective and the training could improve participants knowledge in laboratory techniques in microscopic TB diagnosis.

Medical laboratory accreditation in a resource-limited district health centre laboratory, Addis Ababa, Ethiopia.

BackgroundImproving the quality of medical laboratory services is a high priority in many countries. However, quality management systems for laboratories in resource-limited settings are often inadequate.ObjectivesThis article shares the experiences, benefits and challenges of the laboratory journey towards accreditation in a primary healthcare laboratory in Addis Ababa, Ethiopia.MethodsA retrospective review of laboratory records in Addis Ketema Health Center was conducted from 2012 to 2015. The study was supplemented by observations from some of the authors of this article who worked in the laboratory.ResultsThe laboratory journey towards accreditation began with a baseline assessment in 2012 using the World Health Organization African Region Stepwise Laboratory Quality Improvement Process Towards Accreditation; the baseline score was 78 points (0 stars). After mentorship support, the laboratory improved to 198 points (3 stars) in 2013 and 249 points (5 stars) in 2014. The laboratory scaled up to International Organization for Standardization 15189 requirements and received limited-scope accreditation for tuberculosis sputum microscopy and hematology tests in 2015. After adopting and implementing the standards, steady improvement was observed in the reliability of the laboratory services. Lack of resources was the major challenge the laboratory encountered.ConclusionEven though a remarkable quality performance improvement was observed over the entire process, inadequate skilled personnel was the major challenge identified in the road towards accreditation. Therefore, an appropriate, workload-based staffing structure should be developed to improve and sustain medical laboratory quality standards in resource-limited settings.

Loop-mediated Isothermal Amplification and nested PCR of the Internal Transcribed Spacer (ITS) for Histoplasma capsulatum detection

BackgroundHistoplasmosis is a neglected disease that affects mainly immunocompromised patients, presenting a progressive dissemination pattern and a high mortality rate, mainly due to delayed diagnosis, caused by slow fungal growth in culture. Therefore, a fast, suitable and cost-effective assay is required for the diagnosis of histoplasmosis in resource-limited laboratories. This study aimed to develop and evaluate two new molecular approaches for a more cost-effective diagnosis of histoplasmosis.MethodologySeeking a fast, suitable, sensitive, specific and low-cost molecular detection technique, we developed a new Loop-mediated Isothermal Amplification (LAMP) assay and nested PCR, both targeting the Internal Transcribed Spacer (ITS) multicopy region of Histoplasma capsulatum. The sensitivity was evaluated using 26 bone marrow and 1 whole blood specimens from patients suspected to have histoplasmosis and 5 whole blood samples from healthy subjects. All specimens were evaluated in culture, as a reference standard test, and Hcp100 nPCR, as a molecular reference test. A heparin-containing whole blood sample from a heathy subject was spiked with H. capsulatum cells and directly assayed with no previous DNA extraction.ResultsBoth assays were able to detect down to 1 fg/μL of H. capsulatum DNA, and ITS LAMP results could also be revealed to the naked-eye by adding SYBR green to the reaction tube. In addition, both assays were able to detect all clades of Histoplasma capsulatum cryptic species complex. No cross-reaction with other fungal pathogens was presented. In comparison with Hcp100 nPCR, both assays reached 83% sensitivity and 92% specificity. Furthermore, ITS LAMP assay showed no need for DNA extraction, since it could be directly applied to crude whole blood specimens, with a limit of detection of 10 yeasts/μL.ConclusionITS LAMP and nPCR assays have the potential to be used in conjunction with culture for early diagnosis of progressive disseminated histoplasmosis, allowing earlier, appropriate treatment of the patient. The possibility of applying ITS LAMP, as a direct assay, with no DNA extraction and purification steps, makes it suitable for resource-limited laboratories. However, more studies are necessary to validate ITS LAMP and nPCR as direct assay in other types of clinical specimens.

Development of a colorimetric loop-mediated isothermal amplification assay for rapid and specific detection of Aves polyomavirus 1 from psittacine birds

A colorimetric loop-mediated isothermal amplification (LAMP) assay was developed for the rapid and specific detection of the T gene of Aves polyomavirus 1 (APyV), a causative agent of budgerigar fledgling disease (BFD) in psittacine birds. The amplification can be completed in 40 min at 60 °C, and the results can be visually detected by the naked eye using hydroxyl naphthol blue as a colorimetric indicator. The assay specifically amplified APyV DNA but not other viral and bacterial nucleic acids. The limit of detection of the assay was 5 × 102 DNA copies/reaction, which was comparable to those of previously reported conventional polymerase chain reaction assays. In the clinical evaluation, the LAMP results showed 100% concordance with those of the previously reported PCR assays with regard to specificity, sensitivity, and percentage of overall agreement, with a kappa value of 1.0. These results indicate that the developed LAMP assay will be a valuable tool for the rapid, sensitive and specific detection of APyV from BFD-suspected psittacine bird samples even in resource-limited laboratories.

Rapid detection of Cucumber green mottle mosaic virus in watermelon through a recombinase polymerase amplification assay

Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, is an important quarantine plant virus worldwide, and often causes seriously damages to productions of watermelon, melon, cucumber and other cucurbit crops. In this study, we developed a novel isothermal recombinase polymerase amplification (RPA) technique for detection of CGMMV in watermelon samples. A pair of CGMMV specific RPA primers was prepared based on the conserved CGMMV coat protein gene sequences. The result showed that this RPA detection method can be performed at 38 °C and completed in about 30 min, and there was no cross-reactivity with other common cucurbit viruses. Sensitivity assay showed that this RPA method was more sensitive compared with the regular RT-PCR. Using field-collected watermelon tissue samples, we have demonstrated that this newly developed method is rapid, easy to use and reliable for CGMMV detection, especially in resource-limited laboratories or on-site facilities.

Rapid and equipment-free detection of Phytophthora capsici using lateral flow strip-based recombinase polymerase amplification assay.

Phytophthora capsici is an important oomycete pathogen that causes devastating diseases in various crops. Methods for the rapid detection of P. capsici are important for disease control and eradication programmes. Here, we developed an assay based on lateral flow strip-based recombinase polymerase amplification (LF-RPA) for the rapid and equipment-free detection of P. capsici. The specific primers and a probe were designed using the sequence of Ypt1, and the optimal assay conditions were 40°C for 20min. The specificity of the assay was verified using closely related oomycetes and fungal species, and its detection limit was 10pg of genomic DNA. In combination with a simple DNA extraction method, the LF-RPA assay enabled detection of P. capsici in diseased pepper samples without specialized equipment within 30min. Consequently, the LF-RPA assay developed in this study enables rapid and equipment-free detection of P. capsici and has potential for further development as a diagnostic kit for application in the field or in resource-limited laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: We developed a novel assay based on lateral flow strip-based recombinase polymerase amplification (LF-RPA) for the rapid and equipment-free detection of Phytophthora capsici. In combination with a simple DNA extraction method, the LF-RPA assay detected P. capsici in field samples without specialized equipment within 30min. The assay has potential for further development as a diagnostic kit for application in the field or in resource-limited laboratories.

Body Fluid Testing at John F. Kennedy Medical Center in Liberia.

To apply a simple method to validate testing for albumin, glucose, lactate dehydrogenase (LDH) and total protein (TP) in peritoneal, pleural, and cerebrospinal fluids (CSF) at a hospital in Liberia. Serum and body fluid specimens were mixed to create 100% serum and 25%, 50%, 75%, and 100% fluid tubes, which were tested on a Biotecnica BT3500. Differences less than 10% between calculated and measured concentrations were considered acceptable. The means (confidence intervals) of the percent differences were: albumin/peritoneal 12.8 (6.0-19.7), albumin/pleural 2.8 (1.3-4.2), albumin/CSF 4.8 (2.2-7.5), glucose/peritoneal 4.0 (1.9-6.0), glucose/pleural 4.4 (3.1-5.7), glucose/CSF 2.9 (1.8-4.0), LDH/peritoneal 9.5 (6.3-12.7), LDH/pleural 9.5 (5.4-13.6), LDH/CSF 9.2 (5.2-13.3), TP/peritoneal 7.6 (3.8-11.4), TP/pleural 3.8 (1.5-6.2), and TP/CSF 4.5 (1.0-8.1). All mean differences except for one were less than 10%, allowing for the adoption of clinical testing. The mixing study is a low-cost method for quality-assured testing that can be performed by resource-limited laboratories.

Development of a Standardized Sanger-Based Method for Partial Sequencing and Genotyping of Dengue Viruses.

The global expansion of dengue viruses (DENV-1 to DENV-4) has contributed to the divergence, transmission, and establishment of genetic lineages of epidemiological concern; however, tracking the phylogenetic relationships of these virus is not always possible due to the inability of standardized sequencing procedures in resource-limited public health laboratories. Consequently, public genomic data banks contain inadequate representation of geographical regions and historical periods. In order to improve detection of the DENV-1 to DENV-4 lineages, we report the development of a serotype-specific Sanger-based method standardized to sequence DENV-1 to DENV-4 directly from clinical samples using universal primers that detect most DENV genotypes. The resulting envelope protein coding sequences are analyzed for genotyping with phylogenetic methods. We evaluated the performance of this method by detecting, amplifying, and sequencing 54 contemporary DENV isolates, including 29 clinical samples, representing a variety of genotypes of epidemiological importance and global presence. All specimens were sequenced successfully and phylogenetic reconstructions resulted in the expected genotype classification. To further improve genomic surveillance in regions where dengue is endemic, this method was transferred to 16 public health laboratories in 13 Latin American countries, to date. Our objective is to provide an accessible method that facilitates the integration of genomics with dengue surveillance.

Prevalence and Characterization of Carbapenemase-producing Escherichia coli from a Tertiary Care Hospital in India.

The purpose of this study was to estimate the prevalence and to characterize the carbapenemase-producing Escherichia coli by various phenotypic antimicrobial susceptibility testing methods, and its performance was compared to the gold standard genotypic method. The prevalence of carbapenemase-resistant E. coli was found to be 65%. The phenotypic methods evaluated are cost-effective and can be used in resource-limited laboratories to rule out carbapenem resistance.

Flexible electronic skin with nanostructured interfaces via flipping over electroless deposited metal electrodes

A novel and simple method was developed to quickly pattern and transfer electrodes with nanostructures for fabricating flexible electronic skin (E-skin). A nano/micro-structure embedded Cu electrode can be fabricated from a solution process-based electroless deposition (ELD) on a frosted plastic substrate and subsequently flipped over with an adhesive tape. The fine nano/microstructures on the Cu layer benefit the pressure-electric response of the pressure sensor, demonstrated a high sensitivity: 2.22 kPa−1. This fabricated flexible E-skin can be used for monitoring human physiological signals, such as wrist pulse and thumb bending. This fabrication method is an economical tool for fast prototyping cost-effective wearable electronics for the detection and prediction of diseases. It offers opportunity for researchers from resource-limited laboratories to work on miniaturized wearable devices.

VNTR confirms the heterogeneity of Madurella mycetomatis and is a promising typing tool for this mycetoma causing agent.

The neglected tropical disease mycetoma is a chronic granulomatous inflammatory and infectious disease affecting various body parts. The most common causative agent is the fungus Madurella mycetomatis. In order to study the genetic diversity of this fungus and to monitor any potential outbreaks, a good typing method that can be used in endemic settings is needed. Previous typing methods developed were not discriminative and not easy to perform in resource-limited laboratories. Variable-Number-Tandem-Repeat (VNTR) typing overcomes these difficulties and further enables interlaboratory data comparison. Therefore, in this study we developed a VNTR method for typing M. mycetomatis. Six tandem-repeats were identified in the genome of M. mycetomatis isolate MM55 using an online tandem repeats software. The variation in these repeats was determined by PCR and gel-electrophoresis on DNA obtained from 81M. mycetomatis isolates obtained from patients. These patients originated from Sudan, Mali, Peru, and India. The 81 isolates were divided into 14 genotypes which separated into two main clusters with seven and five subdivisions, respectively. VNTR typing confirms the heterogeneity of M. mycetomatis strains and can be used to study the epidemiology of M. mycetomatis. The results presented in this article are made fully available to the scientific community on request from the Eumycetoma Working Group. We hope that this open resource approach will bridge scientific community working with mycetoma from all around the world and lead to a deeper understanding of M. mycetomatis.

Development of Visual Detection of α-Thalassemia-1 (the – –SEA Deletion) Using pH-Sensitive Loop-Mediated Isothermal Amplification

Detection of α-thalassemia-1 (α-thal-1) carriers provides valuable insight for genetic consulting in prevention and control programs for couples who are at risk of conceiving a fetus with severe thalassemia, both Hb Bart’s hydrops fetalis and hemolytic Hb H disease. The traditional method is complicated, time-consuming and requires high instrument cost and expertise. Loop-mediated isothermal amplification (LAMP) based on pH-sensitive dye technology, shows all the characteristics required of a real-time analysis with simple operation for potential use in the clinical diagnosis of high incidence α-thal-1 [Southeast Asian (SEA) or – –SEA deletion]. Four primers specific for six distinct regions of the α-globin gene deletion were designed and analyzed by LAMP using the pH-indicator dye, phenol red. The amplification of the – –SEA deletion changed the color of phenol red from pink to orange. The diagnostic ability of detection of the – –SEA deletion by pH-sensitive LAMP was validated using both known and unknown blood samples and compared to the conventional polymerase chain reaction (PCR) method. Color inspection of pH-sensitive LAMP products could clearly identify the – –SEA deletion. There was no cross reaction with a normal α-globin gene, α-thal-1 Thai (– –THAI deletion), α-thal-2 [–α3.7 (rightward) and –α4.2 (leftward) deletion] and β-thalassemia (β-thal). Detection of the SEA deletion by pH-sensitive LAMP was consistent as compared to conventional PCR. The pH-sensitive LAMP method developed for this deletion carrier diagnosis has high sensitivity, specificity, simplicity, and requires simple instrumentation that makes it applicable for resource-limited laboratories in rural areas of developing countries.

Comparison of Microbiological Examination by Test Tube and Congo Red Agar Methods to Detect Biofilm Production on Clinical Isolates

Biofilm on medical devices can cause significant diseases and deaths and give a large effecton disease transmission among patients and health providers and potentially increasethe cost of patient treatment. By knowing the presence of biofilm on a patient, one can differentiate the treatment management for that particular patient from the patients without biofilm on their medical device. The purpose of this study was to obtain diagnostic method to detect biofilm formation on isolates from the medical devices by simple method that is easy to do and can be applied in resource-limited microbiology laboratory. 36 specimens obtained from IV Line, CVC, urinary catheter and ETT were grown on Muller Hinton agar and continued with 3 methods, i.e., Test Tube method, Congo Red Agar method and Microtiter Plate Assay method. Results of this study showed Test Tube (nephelometer), Test Tube (visual) and Congo Red Agar in order to have the same sensitivity of 100% but has higher specificity compared to Test Tube method (visual) and Congo Red Agar method in detecting biofilm production on isolates from medical devices that had been plugged into patients body. The biofilm formation inside devices depends on factors, i.e., host, device and the microorganism itself.

Rapid detection of transmissible gastroenteritis virus in swine small intestine samples using real-time reverse transcription recombinase polymerase amplification

A rapid and specific real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA) was developed to detect the transmissible gastroenteritis virus (TGEV) in this study. The primers and exo probe were designed to be specific for a portion of spike (S) gene conserved in TGEV, but absent in the closely related porcine respiratory coronavirus (PRCV). The amplification was performed at 40 °C for 20 min. The assay could only detect the TGEV, and there was no cross-reaction with other pathogens tested. Using the in vitro transcribed TGEV RNA as template, the limit of detection of the developed RT-RPA was 100 copies per reaction. The assay performance was evaluated by testing 76 clinical samples by RT-RPA and a real-time RT-PCR. Fourteen samples were TGEV RNA positive in RT-RPA (18.4%, 14/76), which were also positive in the real-time RT-PCR. The diagnostic agreement between the two assays was 100% (76/76). The R2 value of RT-RPA and real-time RT-PCR was 0.959 by linear regression analysis. The developed RT-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of TGEV in resource-limited diagnostic laboratories and on-site facilities.

Real-time reverse transcription recombinase polymerase amplification assay for rapid detection of porcine epidemic diarrhea virus

Porcine epidemic diarrhea (PED), which is caused by porcine epidemic diarrhea virus (PEDV), is an acute, highly contagious enteric disease characterized by severe watery diarrhea, vomiting, dehydration, and high mortality in suckling piglets. A real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA) was developed based on the nucleocapsid gene of PEDV. RT-RPA assay was performed at 40 °C for 20 min. The assay could detect both the classical and variant PEDV strains, and there was no cross-reaction with other pathogens tested. Using the in vitro transcribed PEDV RNA as template, the analytical sensitivity was 23 copies per reaction. The assay performance was evaluated by testing 76 clinical samples. PEDV RNA positive rate was 55.3% (42/76) by RT-RPA and 59.2% (45/76) by real-time RT-PCR. The diagnostic agreement between the two assays was 96.1% (73/76), and the R2 value of the two assays was 0.903 by linear regression analysis. The developed RT-RPA assay provides a useful alternative tool for simple, rapid and reliable detection of PEDV in resource-limited diagnostic laboratories and on-site facilities.

Evaluation of spoligotyping, SNPs and customised MIRU-VNTR combination for genotyping Mycobacterium tuberculosis clinical isolates in Madagascar.

BackgroundCombining different molecular typing methods for Mycobacterium tuberculosis complex (MTBC) can be a powerful tool for molecular epidemiology-based investigation of TB. However, the current standard method that provides high discriminatory power for such a combination, mycobacterial interspersed repetitive units-variable numbers of tandem repeats typing (MIRU-VNTR), is laborious, time-consuming and often too costly for many resource-limited laboratories. We aimed to evaluate a reduced set of loci for MIRU-VNTR typing in combination with spoligotyping and SNP-typing for routine molecular epidemiology of TB.MethodSpoligotyping and SNP-typing, in combination with the 15 loci MIRU-VNTR typing, were first used to type clinical MTBC isolates (n = 158) from Madagascar. A step by step reduction of MIRU-VNTR loci number was then performed according to the Hunter and Gaston Discriminatory Index (HGDI) and to the Principal component analysis (PCA) correlation with the spoligotype profiles to evaluate the discrimination power inside the generated spoligotype clusters. The 15 MIRU-VNTR was used as reference and SNP-typing was used to determine the main MTBC lineages.ResultsOf the 158 clinical isolates studied, the SNP-typing classified 23 into Lineage 1 (14.6%), 31 into Lineage 2 (19.6%), 23 into Lineage 3 (14.6%) and 81 into Lineage 4 strains (51.3%). 37 different spoligotypes profiles were obtained, 15 of which were unique and 20 in clusters. 15-loci MIRU-VNTR typing revealed 144 different genotypes: 132 isolates had a unique MIRU-VNTR profile and 27 isolates were grouped into 12 clusters. After a stepwise reduction of the MIRU-VNTR loci number within each main spoligotype families, three different sets composed of 5 customised MIRU-VNTR loci had a similar discrimination level to the reference 15 loci MIRU-VNTR in lineage 1, lineage 2 and lineage 3. For lineage 4, a set of 4 and 3 MIRU-VNTR loci were proposed to subtype the Harleem and LAM spoligotype families, respectively. For the T spoligotype family, a set of 5 MIRU-VNTR loci was proposed.ConclusionAccording to the lineages and the spoligotype families, the number of MIRU-VNTR loci can be reduced to get an optimal classification of MTBC. These customized sets of MIRU-VNTR loci reduce workload and save resources while maintaining optimal discriminatory power.

Molecular Serotype-Specific Identification of Non-type b Haemophilus influenzae by Loop-Mediated Isothermal Amplification.

Over the past four decades, the incidence of meningitis caused by Haemophilus influenzae in children has decreased due to widespread vaccination against H. influenzae type b (Hib). The incidence of invasive diseases due to H. influenzae types not included in the vaccines, however, has increased. At present, there are a limited number of diagnostics available to detect non-type b H. influenzae. To address this issue, we developed a rapid, simple, and cost-effective method for detecting serotypes of H. influenzae. We designed LAMP primer sets based on published sequences for H. influenzae capsular types a, c, d, e, and f. The assay was evaluated to determine test reactivity, specificity, and sensitivity. To support its use in patients with suspected meningitis, we evaluated the detection limit of the non-Hib serotype specific LAMP assay using bacterial genomic DNA-spiked cerebrospinal fluid (CSF) specimens. The reactivity and specificity of the LAMP assays were confirmed using six serotypes and non-typeable H. influenzae strains, plus eight strains of other Haemophilus species and non-Haemophilus genera. The detection limits of the LAMP assay for capsular types a, c, d, e, and f were 102, 102, 102, 103, and 10 copies per reaction, while those of the PCR assay were 104, 104, 103, 103, and 104 genome copies per reaction, respectively. Using DNA-spiked CSF specimens, the detection limit of the LAMP assay was equivalent to that using purified DNA as the template. However, the detection limit of the PCR was reduced from 103 to 104 genome copies per reaction for serotype d and from 103 to 105 genome copies per reaction for serotype e. To the best of our knowledge, this is the first report of a serotype-specific identification assay for H. influenzae using the LAMP method. Our results suggest the potential of LAMP methods for patients with suspected meningitis in resource-limited laboratories or public health surveillance systems.

Fast and low-cost patterning of electrodes on versatile 2D and 3D substrates by cutting and origami cohesive thermoplastic for biosensing applications

In this work, cohesive thermoplastic-assisted patterning was successfully used to fabricate versatile 2D and 3D electrodes for biosensing application. The cohesive thermoplastic template was fabricated by cutter shaping to remove wet-chemical processes and UV-lithography. This low cost approach is compatible with versatile inks and substrates. Both single and multi-step grafting of inks work well with cohesive thermoplastic templates to form electronic units on substrates. Other than work on a 2D flat substrate, the cohesive thermoplastic origami can easily fabricate electrodes on 3D objects, demonstrating itself as a good candidate to implant electronic unit on a stereotype object. The flexibility, convenience and operability seen here make this method a fast prototype method to evaluate the proof-of-concept design. It also provides alternative fabrication solutions for researchers from resource-limited laboratories to work on miniaturized devices.

A simple laboratory algorithm for diagnosis of melioidosis in resource-constrained areas: a study from north-central Vietnam

ObjectivesMelioidosis may be endemic in many tropical developing countries, but diagnosis of the disease is currently unreliable in resource-limited areas. We aimed to validate a simple and cheap laboratory algorithm for the identification of Burkholderia pseudomallei from clinical specimens in parts of Vietnam where the disease has not previously been reported. MethodsIn June 2015, we conducted training courses at five general hospitals in north-central provinces in order to raise awareness of the disease and to introduce a simple and cheap laboratory identification algorithm for B. pseudomallei including the three-antibiotic disc test. ResultsUntil the end of the year (7 months later), 94 suspected B. pseudomallei strains resistant to gentamicin and colistin but sensitive to amoxicillin/clavulanic acid were detected in clinical specimens from 70 patients. All strains were further confirmed as B. pseudomallei by using a specific TTSS1 real-time PCR assay and recA sequencing analysis. Among positive blood cultures, positive rates with B. pseudomallei ranged from 3.4% (5/147) to 10.2% (32/312) in the various clinics. A total of 82.8% (58/70) patients were bacteraemic, with a mortality of 50% (18/36) among patients with known outcome. No death occurred in nonbacteraemic patients. ConclusionsOur results demonstrate that the introduction of a simple and easy-to-perform laboratory algorithm for the identification of B. pseudomallei from clinical samples, together with clinical awareness raising, can lead to the diagnosis of a significant number of melioidosis cases in resource-limited clinical laboratories which previously did not identify the pathogen.