Role of the Laboratory in the Diagnosis of Poxvirus Infections.

Although WHO-led global efforts led to eradication of smallpox over four decades ago, other poxviruses, especially monkeypox, have re-emerged to occupy the ecological niche vacated by smallpox. Many of these viruses produce similar lesions thus mandating a prompt laboratory confirmation. There has been considerable evolution in the techniques available to diagnose these infections and differentiate between them. With the 2022 multi-country outbreak of monkeypox, significant efforts were made to apprise the laboratory diagnosis of the virus and numerous real-time-PCR-based assays were made commercially available. This chapter discusses the sample collection and biosafety aspects along with the repertoire of diagnostic modalities, both traditional and emerging, for poxviruses which a special focus on monkeypox. The advantages and disadvantages of each technique have been illustrated. We have also reflected upon the newer advances and the existing lacunae.

Impact of relative humidity on SARS-CoV-2 RNA extraction using Nextractor automated extraction system.

This study investigated the influence of relative humidity (RH) on the efficiency of SARS-CoV-2 RNA extraction using the Nextractor automated system. Experiments employing clinical samples demonstrated satisfactory sensitivity and reproducibility for RNA extraction at low humidity (below 50% RH). Conversely, extractions at high humidity (above 70% RH) resulted in complete failure of reverse transcription-polymerase chain reaction assays, with neither SARS-CoV-2 RNA nor the human RNase P gene (internal control) detected. Analysis suggested that residual ethanol, incompletely evaporating due to high humidity, acted as a potent polymerase chain reaction inhibitor in these samples. These findings highlighted the importance of maintaining optimal laboratory humidity (<50% RH) for reliable SARS-CoV-2 RNA extraction using the Nextractor system. Furthermore, laboratories should implement strategies such as regular humidity monitoring, staff training on humidity's impact, and system validation under specific humidity conditions to ensure accurate molecular diagnostic workflows for COVID-19 testing.

Strategies to Decrease the Prevalence of Soil-Transmitted Helminths in Central India.

Background Intestinal parasites are a major public health problem in tropical countries. Over 1.5 billion people are infected with soil-transmitted helminths (STH), of which 225 million are in India. Parasitic infections are associated with poor sanitation, lack of safe potable water, and improper hygiene. Materials and Methods The study was undertaken to ascertain the impact of control strategies, namely open-defecation free drive and mass drug administration of single dose albendazole. Stool samples received at AIIMS Bhopal Microbiology laboratory, across all age groups, were studied for protozoan trophozoites/cysts and helminthic ova. Results Out of 4,620 stool samples, 389 (8.41%) were positive either for protozoal or helminthic infections. Protozoan infections were more common than helminthic infections with Giardia duodenalis infection being the most common, 201 (51.67%), followed by Entamoeba histolytica , 174 (44.73%). The helminthic infections constituted 14 (3.5%) of the positive stool samples with Hookworm ova in 6 (1.5%) cases. Conclusion This study proves that strategies, namely "Swachh Bharat Abhiyan" and "National Deworming Day" started in 2014 and 2015 led to significant reduction of intestinal parasite infections in Central India, with a higher reduction of STH compared with protozoan parasite infection being ascribed to the activity spectrum of albendazole.

Performance Evaluation of Different RT-PCR Kits for the Direct Detection of SARS-CoV-2 in Preheated Specimens.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created high demand for molecular kits and consumables for mass screening of suspected individuals. Direct real-time polymerase chain reaction (RT-PCR) assay without nucleic acid extraction has several advantages in saving testing time and cost and helps in the rapid reporting of SARS-CoV-2. The present study evaluated the analytical performance of four SARS-CoV-2 RT-PCR for direct RT-PCR testing using preheated specimens. Methods A total of 100 clinical specimens were selected and divided into three different groups: (1) group I: 20 SARS-CoV-2 positive specimens with high viral load, viz., low Ct values (< 30 Ct), (2) group II: 50 SARS-CoV-2 positive specimens with low viral load, viz., high Ct values (> 30 Ct), and (3) group III: 30 SARS-CoV-2 negative specimens. Specimens were heat-inactivated at 70°C for 10 minutes and cooled down at 4°C and were evaluated for standard and direct RT-PCR method by using ViralDtect-II Multiplex Real-Time PCR kit, TaqPath COVID-19 Combo kit, COVIDsure Pro Multiplex RT-PCR kit, and Hi-PCR Coronavirus (COVID-19) Multiplex Probe PCR kit. Results Results showed that except ViralDtect-II kit, the other three TaqPath COVID-19 Combo kit, COVIDsure Pro kit, and Hi-PCR Coronavirus (COVID-19) RT-PCR kit were able to amplify all the SARS-CoV-2 genes in the direct RT-PCR method using preheated specimens. In group I specimens, 100% sensitivity was observed in all three RT-PCR kits. In group II specimens, COVIDsure Pro kit was found to be superior among other kits. Conclusion Direct RT-PCR method during pandemic situation is valuable and cost effective for the detection of SARS-CoV-2. All three TaqPath COVID-19 Combo kit, COVIDsure Pro kit, and Hi-PCR Coronavirus (COVID-19) RT-PCR kit can be used for direct RT-PCR method and COVIDsure Pro kit performance was found to be superior among all.

Intraoperative Surgical Wound Contamination May Not Lead to Surgical-Site Infection in Patients Undergoing Clean Orthopaedic Procedures.

Objectives Surgical-site infections (SSIs) can complicate virtually any surgical procedure. While SSI can result from numerous causes, contamination of the surgical field can also contribute to it. Intraoperative bacterial contamination during clean orthopaedic procedures can be detected using perioperative cultures. We hypothesized that perioperative cultures could be used to predict possibility of development of SSI in patients undergoing clean orthopaedic surgeries. Materials and Methods We conducted a prospective cohort study at a tertiary care hospital over a 2-year period. Intraoperative surgical wound lavage fluid and closed suction drain tip obtained in the postoperative period were sent for aerobic culture. All patients were followed up to look for the development of SSI for a period of at least 30 days for those undergoing nonimplant surgery, and 90 days for those with implant surgery. Statistical Analysis Means with standard deviation of the continuous data were calculated. Fisher's exact test and chi-square test were used for the analysis of the categorical variables. Relative risk and odds ratio were calculated to evaluate the association of the parameters under study with SSI. Results A total of 384 patients satisfying the inclusion and exclusion criteria were included. Perioperative cultures detected surgical wound contamination in 39 patients (10.1%). Forty-five patients (11.7%) developed SSI during the follow-up period. Skin commensals constituted 59% of perioperative contaminants and accounted for 20% of the SSIs. The relative risk of developing SSI with perioperative contamination was 0.41 (95% confidence interval: 0.09-1.63). Conclusion Intraoperative surgical-site contaminants could be detected using perioperative cultures. However, these contaminants did not lead to SSI. Timely treatment of perioperative contamination with appropriate antibiotics and local wound care probably helped in the reduction of SSI.

A Pilot Study on Carbapenemase Detection: Do We See the Same Level of Agreement as with the CLSI Observations.

INTRODUCTION: Rapid identification of carbapenemase producing organisms is of great importance for timely detection, treatment and implementation of control measures to prevent the spread. The Modified Hodge Test (MHT) and Carba NP test is recommended by CLSI for the detection of carbapenemases in Enterobacteriaceae. However, MHT may give false positive results or fail to detect metallo ß-lactamases (MBLs). In the US, MHT is the most widely used test for detection of carbapenemases and has been found to have a sensitivity and specificity of >90% for bla KPC producers. However, in India, the prevalence of bla NDM is higher than bla KPC producers. AIM: To evaluate the usefulness of CarbaNP in an Indian setting. MATERIALS AND METHODS: A total of 260 isolates of carbapenem resistant E.coli (n=57), Klebsiella spp. (n=85), Pseudomonas aeruginosa (n=60), and Acinetobacter baumannii (58) isolated from clinical specimens between 2012-2014 at the Christian Medical College, Vellore were included in the study. All the carbapenem resistant isolates were subjected to CarbaNP, MHT and multiplex PCR for detection of carbapenemase genes. RESULTS: CarbaNP was found to be positive in 88% (n=50/57), 81% (n=69/51), 38% (n=23/60) and 81% (n=47/58) for E.coli, Klebsiella spp., P. aeruginosa, and A. baumannii respectively. While in MHT it showed, 89% (n=51/57) and 81 % (n=69/85) for E.coli and Klebsiella spp. respectively. In P.aeruginosa, synergy testing of imipenem plus cloxacillin showed that, 65% of CarbaNP negatives were ampC producers. Overall, the sensitivity and specificity of CarbaNP was found to be 94% and 100 for bla NDM; 77% and 100 % for bla OXA-48 like producers and 81% and 100% for CarbAcinetoNP respectively. CONCLUSION: This observation was more than what was reported in CLSI guidelines. Therefore, it is advisable to evaluate an assay for better laboratory diagnosis at respective regions.