Performance evaluation of Truenat™ Beta CoV & Truenat™ SARS-CoV-2 point-of-care assays for coronavirus disease 2019.

Background & objectives: The rapid diagnosis of coronavirus disease 2019 (COVID-19) is a significant step towards the containment of the virus. The surge of COVID-19 cases in India and across the globe necessitates a rapid and sensitive molecular assay. Rapid point-of-care (PoC) assays (Truenat Beta CoV and Truenat SARS-CoV-2 assays) for the diagnosis of COVID-19 have been developed which are expected to shorten the turnaround time of reporting of results and also can be used for field investigations of COVID-19. The objectives of the study were to validate the performance of Truenat Beta CoV and Truenat SARS-CoV-2 PoC assays for the detection of SARS-CoV-2 infected cases with reference to analytical sensitivity, precision/inter-machine variation, clinical sensitivity and clinical specificity. Methods: The rapid PoC screening and confirmatory assays were prospectively validated at the State Level Virus Research and Diagnostic Laboratory at Bangalore Medical College and Research Institute, Bengaluru, under technical supervision by the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune. Real-time reverse transcription-polymerase chain reaction (rRT-PCR)was considered as the reference standard against which the rapid assays were validated for all samples tested based on analytical sensitivity, precision/inter-machine variation, clinical sensitivity and clinical specificity. Results: Truenat Beta CoV and Truenat SARS-CoV-2 assays showed concordant results when compared with the reference standard rRT-PCR. These PoC assays exhibited 100 per cent sensitivity, specificity, positive predictive value and negative predictive value. Interpretation & conclusions: Truenat Beta CoV and Truenat SARS-CoV-2 assays showed concordance with the reference standard assay and may be recommended for screening and confirmation of SARS-CoV-2 in the field settings.

First detection of Varicella Zoster Virus clade 9 cases in India during mpox surveillance.

BACKGROUND: The multi-country mpox outbreak across the globe has led to the systematic surveillance of mpox cases in India. During the surveillance of mpox, we encountered cases of Varicella Zoster Virus (VZV) in suspected mpox cases amongst children & adults. This study focused on the genomic characterization of VZV in India. METHODS: A total of 331 mpox suspected cases were tested for VZV through real-time PCR, and the positive samples were subjected to next-generation sequencing to retrieve the whole genome of VZV using CLC genomics software. Phylogenetic analysis has been done in MEGA 11.0 software to identify circulating clades. RESULT: Of the 331 suspected cases, 28 cases with vesicular rashes were found to be positive for VZV. The maximum genome could be retrieved from the clinical specimens of 16 cases with coverage greater than 98% when mapped with reference strain Dumas (NC 001348). The phylogenetic analyses of these sequences determined the circulation of clades 1, 5, and 9 in India. Further, the sequence analysis demonstrated non-synonymous single nucleotide polymorphism (SNPs) among specific ORF of VZV including ORF 14, ORF 22, ORF 36, ORF 37 and ORF 51. Although clade 1 and 5 has been reported earlier, the circulation of clade 9 of VZV has been determined for the first time in India. CONCLUSION: Although the circulation of different clades of VZV was reported from India, the presence of clade 9 was detected for the first time during the mpox surveillance.

Whole-Genome Sequencing of SARS-CoV-2 Strains from Asymptomatic Individuals in India.

We performed targeted genome sequencing of 11 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples collected from asymptomatic individuals. These nasopharyngeal and oropharyngeal samples were collected during the first wave of coronavirus disease 2019 (COVID-19) in Karnataka, India. Nine strains were found to be Nextstrain clade 20B (PANGO lineage B.1.1.519, GISAID clade GR), and two were identified as clade 20A (PANGO lineage B.1.619, GISAID clade G). The spike protein mutation D614G was observed across all the sequenced strains.

Detection of SARS-CoV-2 in Clinical Samples: Target-specific Analysis of Qualitative Reverse Transcription-Polymerase Chain Reaction(RT-PCR) Diagnostic Kits.

INTRODUCTION: The Coronavirus disease 2019 pandemic caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the rise of many available modalities for diagnosis. One such modality is the Reverse Transcription-Polymerase Chain Reaction (RT-PCR) kits which require evaluation amongst the many available commercial kits in the market. METHODS: We conducted a performance evaluation of twelve RT-PCR SARS-CoV-2 commercial kits. A total of 75 nasopharyngeal and oropharyngeal clinical samples were selected with their cycling threshold (Ct) values. Inclusion of 5 gene targets: E gene, N gene, S gene, RdRp and ORF1ab were assessed. Data was analyzed using R software version 4.1.1 and Microsoft Excel. RESULTS: We observe that, the positive sample's Ct values differs significantly across the 12 diagnostic kits. However, for gene-specific analysis, we observe that, positive sample's Ct values does not differ significantly across gene targets. There is significant difference in Ct values in Commercial kits targeting all genes except S-gene. All the commercial kits Altona (E and S genes), Thermo (ORF1ab and N genes), Multiplex (E, ORF1ab, RdRdp genes), Meril (N and ORF1ab genes), S D Biosensor (E and ORF1ab genes), Lab Gun (RdRp and N genes) and Lab systems (ORF1ab and E genes) scored a sensitivity of 100%. All other kits scored sensitivity above 95% and lowest sensitivity with the Genes2me (E gene) and Genes2me (RdRp) at 95.08% each. All kits were 100% specific. CONCLUSION: This study provides an accurate comprehensive assessment of the different kits in the detection of SARS-CoV-2 which may promote standardization of testing across laboratories.

Clinical and Molecular facets of Dengue Virus infection from Bengaluru, South India.

BACKGROUND: Dengue virus (DENV) continues to be an epidemic with high mortality rates. The clinical features, especially in the early phase of infection, are nonspecific and there is no single marker that can be reliably deployed for diagnostics. Further, serotype and genotype diversity is not clearly understood. This study was conceived to understand the performance characteristics of various diagnostic markers; serotype and genotype distribution is thus a vital requirement. METHODS: A subset of blood samples was obtained for all the clinically suspected Dengue cases during the period January to December 2017. The samples were tested for IgM and IgG antibodies and NS1 antigen by both ELISA and rapid tests. Real-time PCR, Conventional PCR and sequencing was performed based on the serology results. Correlation of the data with demographic and clinical details was used to analyze the performance characteristics of various tests. RESULTS: Clinical signs and symptoms could not predict dengue positivity due to lack of specific symptoms. The performance of IgM rapid test was found to be lower than the ELISA method (53.5% agreement). The NS1 rapid and NS1 ELISA tests were comparable (89.2% agreement). Majority of the infections were caused due to DEN-2 serotype and phylogenetic analysis revealed all the sequenced DEN-2 serotypes belong to Genotype IV. Three sequences were deposited into NCBI GenBank (GenBank accession number MW583116, MW579054 and MW579053). CONCLUSION: Our comprehensive data suggests that NS1 ELISA and PCR are best used in the early phase of dengue infection (< 5 days post-onset of fever), whereas IgM antibody detection is reliable only in the late phase. We also highlight the unreliable performance of rapid tests.

Low rate of gut colonization by extended-spectrum ß-lactamase producing Enterobacteriaceae in HIV infected persons as compared to healthy individuals in Nepal.

A worldwide increase in the gastrointestinal colonization by extended-spectrum ß-lactamase (ESBL)-producing bacteria has been observed. Their prevalence amongst Healthy People Living with HIV (HPLWH) has not been investigated adequately. The aim of this study was to determine and compare the rates of and risk factors for intestinal carriage and acquisition of extended-spectrum ß-lactamase producing Enterobacteriaceae (ESBL-E) and carbapenemase-producing Enterobacteriaceae (CPE) among healthy people living with HIV (HPLWH) and healthy HIV negative population in the community. A cross-sectional study was conducted. Rectal swabs from HPLWH (n = 119) and HIV negative individuals (n = 357) from the community were screened for ESBL and CPE. Phenotypically confirmed ESBL-E strains were genotyped by multiplex PCR. The risk factors associated with ESBL-E colonization were analyzed by a multivariable conditional logistic regression analysis. Specimen from 357 healthy volunteers (213 female and 144 male) and 119 HPLWH (82 female and 37 male) with a median age of 30 [IQR 11-50] years were included in the study. ESBL colonization were found in 45 (37.82% [CI 29.09, 47.16]) and 246 (68.91% [CI 63.93, 73.49]), HPLWH and healthy HIV negative participants respectively. HPLWH had lower ESBL carriage rate (odds ratio 0.274 [CI 0.178, 0.423]) compared to healthy HIV negative subject's (p<0.01). In this study, no carbapenemase-producing bacteria were isolated.CTX-M-15 type was the most predominant genotype in both groups. Livestock contact and over-the-counter medications were significantly associated with a higher ESBL-E carriage rate among healthy subjects. This is the first study in Nepal that has demonstrated a high rate of gut colonization by ESBL-E in the community, predominantly of blaCTX-M-15 genotype. This study divulges the low fecal carriage rate of ESBL producing bacteria in HPLWH group compared to healthy individuals in western Nepal. The factors responsible for this inverse relationship of HIV status and gut colonization by ESBL-E are unidentified and require further large-scale study.

Neonatal sepsis due to glycopeptide resistant Enterococcus faecium from colonized maternal gut- rare case evidence.

Background: Vancomycin-resistant enterococcal infections in the neonatal ICU are growing global problems. We report a case of neonatal septicemia by multidrug-resistant vancomycin-resistant Enterococcus faecium (VRE), the source of infection being the mother's gut. Case presentation: A newborn male child admitted to the neonatal intensive care unit (NICU) was diagnosed to have mild meconium aspiration syndrome, early onset neonatal septicemia, and bacteremia by multidrug and vancomycin-resistant Enterococcus faecium. Screening of gut flora of the baby and the mother were carried out to trace the source of infection. Stool cultures of the mother and the baby yielded Vancomycin-Resistant Enterococcus faecium. All three isolates of Enterococcus faecium had similar antibiogram, harbored the vanA gene and similar pulsed-field gel electrophoresis pattern. Baby responded to the 1 week therapy with oral linezolid suspension 20 mg/kg/day, 1 ml/t.d.s. No VRE was isolated from baby on a repeat stool culture 1 week after the linezolid therapy. He was discharged with the advice for the continuance of linezolid for seven more days. Conclusion: Isolation of MDR-VRE from the blood culture of the baby and stool specimens of the mother and the baby with the same antibiogram profile and clonal similarities reveals that maternal gut colonization was responsible for neonatal sepsis. Optimal infection control measures and the development of guidelines for monitoring VRE colonization in pregnant women might be useful in reducing the occurrence of neonatal sepsis.

Detection of Blastocystis in clinical stool specimens using three different methods and morphological examination in Jones' medium.

BACKGROUND: Blastocystis was identified almost a century ago, yet its biology and pathogenicity status in humans is obscure. Studies on Blastocystis in India are scanty and are mostly microscopy based. This study compared three detection modalities to determine their efficiency in the identification of Blastocystis in human feces. MATERIALS AND METHODS: A total of 279 stool samples were screened using microscopy, culture (Jones' medium), and polymerase chain reaction (PCR)-based methods. Among the three, PCR is considered the gold standard test for detection of Blastocystis, as it helps to authenticate the sensitivity, specificity, and kappa agreement obtained by the other two tests. The morphological features of Blastocystis were recorded at 24, 48, and 72 h. After positive morphological identification, ten samples were cultured on Löwenstein-Jensen (LJ) medium and Locke's egg slant medium. RESULTS: The sensitivity and specificity determined on the basis of microscopy were 36.2% and 99.4%, respectively. On the other hand, Jones' medium showed 67.6% sensitivity and 100% specificity. Further, we documented various morphological and reproductive features of Blastocystis using various staining techniques on cultures positive in Jones' medium. In addition, we also found that LJ medium was not equally efficacious as Jones' medium in assisting the growth of Blastocystis. CONCLUSIONS: Although molecular diagnosis is a necessary tool for understanding the true epidemiology of Blastocystis, in laboratories devoid of molecular detection facilities, stool microscopy in conjunction with stool culture on Jones' medium could serve as the best alternative tool for the detection of Blastocystis.

Severe Blastocystis subtype 3 infection in a patient with colorectal cancer.

Colorectal cancer (CRC), more of lifestyle-related disorder, is one of the deadliest types of cancer across the globe. Nevertheless, infectious agents could be responsible for 20% of cancer. Recent findings have indicated the association of Blastocystis in CRC and recommend routine screening for Blastocystis. Herein, we describe a case of CRC with severe Blastocystis infection.

Evaluation of the utility of conventional polymerase chain reaction for detection and species differentiation in human hookworm infections.

BACKGROUND: Human hookworm infection is caused mainly by Necator americanus and Ancylostoma duodenale. Among the zoonotic hookworm species, only Ancylostoma ceylanicum causes potent human infections where dogs and cats act as reservoir of infection. Hence, species differentiation is imperative because the eradication of both anthroponotic and zoonotic hookworm depends on the concurrent human and animal health programs, hygienic practices, and mass drug administration for humans and dogs. OBJECTIVE: This study was performed to evaluate the utility of polymerase chain reaction (PCR) for detection of hookworm infections. MATERIALS AND METHODS: A total of 209 stool samples were collected and subjected to stool microscopy, Kato-Katz method to identify the intensity of the infection, coproculture for L3 larval identification and species differentiation and semi-nested PCR with sequencing. RESULTS: The prevalence of hookworm was estimated as 7.6%. Highest hookworm prevalence was seen in 20-30 years of age group. Majority of the infections were mild intensity infections. Sensitivity of stool microscopy was found to be 81.2% and the specificity was 100%. Sensitivity of Kato-Katz method was 87.5% and specificity was 100%. True positivity by agar plate culture was 83.3% and false positivity rate was 16.6%. CONCLUSION: Stool microscopy is the major mode of detection, but it has a higher false negative rate. Coproculture is time-consuming and needs the expertise to differentiate the species. On the other hand, PCR is known to be a sensitive, specific, and a reliable investigative tool which can help in diagnosis as well as in species differentiation.

Subdural haematoma in Plasmodium falciparum and Plasmodium vivax mixed infection presenting multiple clinical complications.

A 40-year-old man was admitted to hospital with a 5 day history of fever, restlessness and altered sensorium. Peripheral blood smears showed a Plasmodium vivax and Plasmodium falciparum mixed infection as revealed by the presence of rings, schizonts and gametocyte forms of the parasites. The patient soon became unconscious due to subdural haematoma (SDH) associated with disseminated intravascular coagulation and thrombocytopenia. Immediate intervention with a right fronto-parieto temporal craniectomy, evacuation of the SDH and intravenous quinine administration resulted in the patient's complete recovery within 8 days of admission, and he was discharged in good clinical condition.

Co-infection with hepatitis viruses among HIV-infected individuals at a tertiary care centre in South India.

We studied the occurrence of hepatitis B (HBV) and hepatitis C (HCV) virus co-infections in human immunodeficiency virus (HIV)-infected individuals in a tertiary care centre. The occurrence of co-infections in our study was less than in the literature reported from the West. However, routine screening of these co-infections in HIV patients is recommended to avoid possible liver-related morbidity and mortality as a part of improving their quality of life.