Application of Polymerase Chain Reaction in Diphtheria Laboratory Examination: A Field Need

Background: Indonesia is one of the five countries with the highest number of diphtheria cases worldwide. Diphtheria is caused by the toxigenic strains Corynebacterium diphtheriae, C. ulcerans, and C. pseudotuberculosis. The diphtheria-causing bacteria can be identified using conventional and molecular methods, including polymerase chain reaction (PCR) assay. We used the PCR assay as additional testing, because in island countries like Indonesia, specimen transport is a serious challenge to maintaining bacterial survival. Objectives: This study aimed to evaluate the PCR assay as additional testing to identify diphtheria-causing bacteria in the diphtheria laboratory. Methods: In this cross-sectional study, a total of 178 pairs of the throat and nasal swabs from diphtheria suspected cases and close contacts were collected from seven provinces in Indonesia in 2016. All samples were directly identified by the conventional method and multiplex PCR assay. Statistical analysis was conducted using the 2 × 2 tables to determine the sensitivity and specificity of both methods, while the χ2 test was used to examine the correlation between specimen examination delay and the differentiation of results. A P-value < 0.05 was considered as statistically significant. Results: Out of 178 examined samples, eight samples were identified as diphtheria-positive by both the conventional method and PCR assay, while nine samples were only detected by the PCR assay. All diphtheria-causing bacteria found in the positive samples were toxigenic C. diphtheriae. The diphtheria-causing bacteria were found in 27.6% of cases and 6.0% of close contacts using the PCR assay versus 13.8% of cases and 2.7% of close contacts using the conventional method. Statistical analysis showed that the PCR assay is about twice more sensitive than the conventional method. There was a significant correlation between the differentiation of results and > 72 hours’ specimen examination delay with a P-value of 0.04 (< 0.05). Conclusions: The PCR assay is more sensitive than the conventional method to identify diphtheria-causing bacteria and may be applied as additional testing to increase the positivity rate of diphtheria-confirmed cases in Indonesia as an archipelago country where geographical factors and specimen transport are real obstacles.

Evaluation of Transport Media and Specimen Transport Conditions for the Detection of SARS-CoV-2 by Use of Real-Time Reverse Transcription-PCR

ABSTRACT The global coronavirus (CoV) disease 2019 (COVID-19) pandemic has resulted in a worldwide shortage of viral transport media and raised questions about specimen stability. The objective of this study was to determine the stability of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) RNA in specimen transport media under various storage conditions. Transport media tested included UTM, UTM-RT, ESwab, M4, and saline (0.9% NaCl). Specimen types tested included nasopharyngeal/oropharyngeal swabs in the above-named transport media, bronchoalveolar lavage (BAL) fluid, and sputum. A high-titer SARS-CoV-2 remnant patient specimen was spiked into pooled SARS-CoV-2 RNA-negative specimen remnants for the various medium types. Aliquots of samples were stored at 18°C to 26°C, 2°C to 8°C, and −10°C to −30°C and then tested at time points up to 14 days. Specimens consistently yielded amplifiable RNA with mean cycle threshold differences of <3 over the various conditions assayed, thus supporting the use and transport of alternative collection media and specimen types under a variety of temperature storage conditions.

Effect of Specimen Transport Network System in the Turnaround Time on Patients Tuberculosis Genexpert Test Results in the West Region of Cameroon

Introduction : Specimen transport network system is a major tool in getting quick turnaround time for patient results in general and for tuberculosis (TB) diagnosis in particular given that TB is an airborne disease and any mismanagement of its specimens, sputum in particular, can lead to generation of aerosols, hence spread and infection of the persons transporting the specimens and the health care personnel receiving the specimens and the delay in the diagnosis leading to more spread of the bacilli and up to the death of the patient. It is therefore of great essence to establish a reliable specimen transport network system which is essential for effective TB patient care, allowing for rapid diagnosis, initiation of treatment and patient follow up in the West Region of Cameroon and Cameroon as a whole. This article is therefore aimed at establishing a better specimen transport network system to better the turn- around time for TB specimens in the West Region of Cameroon. Method: A descriptive retrospective study by means of secondary data collection was carried from January 2016 to July 2017 on 1,130 specimens requested by clinicians before the GeneXpert instruments were introduced in the West Region of Cameroon and from October 2017 to July 2018 when two GeneXpert instruments were introduced in the West region. Request forms with date of test requested at the health facilities in the West region and results registers in which date results from the Reference Laboratory were communicated to health facility in the West Region, were used as the data sources to calculate the turn- around time (TAT). TAT was analyzed in hours and converted to days using excel. Results : Results from this study shows an average decrease in turnaround time of patient’s results from 26 days when the specimens were tested only at the Reference laboratory in Bamenda to 7 days when two GeneXpert instruments were introduced in the West region with a total of 2 platforms where tests could possibly be done. Conclusion : Re designing a better specimen transport network system and increasing the number of Xpert platforms not only in the West region but in all other regions of Cameroon, will reduce TAT for TB results to only 01 to 02 days, thus increase quality of prevention and treatment programs, thereby reducing costs and live savings of the population.

Laboratory

This chapter outlines how clinical management decisions in humanitarian emergency situations can be greatly enhanced by a well-functioning laboratory. It provides the fundamentals of diagnostic services, including quality assurance and specimen transport, and outlines the basic procedures for the most common tests.