A Comparative Analysis of Microscopy, Coproantigen Serology, and Nested Multiplex PCR in the Laboratory Diagnosis of Entamoeba histolytica Infection.

Objectives Amoebiasis is caused by the most common intestinal protozoan parasite Entamoeba histolytica . This parasite causes amoebic colitis, which is manifested by diarrhea, followed by dysentery. The laboratory diagnosis of intestinal amoebiasis in most cases is by microscopic examination of stool samples. Other nonroutine methods include coproantigen enzyme-linked immunosorbent assay (ELISA) from stool samples, serum ELISA for antibodies, stool culture, isoenzyme analysis, and polymerase chain reaction (PCR). The present study aimed to comparatively analyze the different diagnostic modalities used for the detection of E. histolytica from the stool sample of patients with intestinal amoebiasis. Materials and Methods This study was undertaken with 631 patients, during a period of 3 years, from January 2017 to December 2019. Stool specimen obtained from each patient was subjected to direct microscopic wet mount examination, coproantigen ELISA, and nested multiplex PCR, respectively. Results Out of all the patients tested, 5.2% were positive for E. histolytica. Among the positive cases, stool microscopy was positive in 3.17%, coproantigen ELISA was positive in 29 (4.6%) cases, and PCR was positive in 30 (4.75%) cases. Statistical Analysis The prevalence of E. histolytica infection was summarized as percentages. The three diagnostic tests done were statistically analyzed, taking microscopy as the gold standard. The agreement between techniques (microscopy, coproantigen ELISA, and PCR) was analyzed with kappa statistics. Sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were summarized as percentage with 95% confidence interval. Conclusion In all suspected amoebiasis cases, a combination of stool microscopy, coproantigen testing with molecular detection of the parasite offers the best approach to diagnosis of this parasitic infection.

Clinical profile and outcomes of multisystem inflammatory syndrome in children (MIS-C): Hospital-based prospective observational study from a tertiary care hospital in South India.

AIM: To study the clinical profile and outcomes in children with multisystem inflammatory syndrome in children (MIS-C). METHODS: Children aged 1 month to 15 years presenting with MIS-C (May 2020 to November 2021) were enrolled. Clinical, laboratory, echocardiography parameters and outcomes were analysed. RESULTS: Eighty-one children (median age 60 months (24-100)) were enrolled. Median duration of fever was 5 days (3-7). Twenty-nine (35.8%) had shock (severe MIS-C) including 23 (28.3%) requiring inotropes (median duration = 25 h (7.5-33)). Ten required mechanical ventilation, 12 had acute kidney injury and 1 child died. Left ventricular (LV) dysfunction was seen in 38 (46.9%), 16 (19.7%) had coronary artery abnormalities (CAA) and 13 (20%) had macrophage activation syndrome. Sixty-one (75.3%) were SARS CoV-2 positive (10 by RT-PCR and 51 by serology). Sixty-eight (83.9%) received immunomodulators. Younger age was significantly associated with CAA (P value = 0.05). Older age, LV dysfunction, SARS CoV-2 positivity, low platelet count and elevated serum ferritin were significantly associated with severe MIS-C (univariate analysis). Younger age was an independent predictor of CAA (P = 0.05); older age (P = 0.043) and low platelet count (P = 0.032) were independent predictors of severe MIS-C (multivariate logistic regression analysis). CONCLUSION: Our patients had diverse clinical manifestations with a good outcome. Younger age was significantly associated with CAA. Older age, LV dysfunction, low platelet count and elevated serum ferritin were significantly associated with severe MIS-C. Younger age is an independent predictor of CAA. Older age and low platelet count are independent predictors of severe MIS-C.

Prevalence and speciation of non-tuberculous mycobacteria among pulmonary and extrapulmonary tuberculosis suspects in South India.

BACKGROUND AND OBJECTIVES: Non tuberculous mycobacteria (NTM) is an emerging opportunistic pathogen increasing globally and indistinguishable from tuberculosis (TB), which remains a challenge particularly in developing countries. This study aimed to identify the prevalence and diversity of NTM among both pulmonary TB (PTB) and extrpulmonary TB (EPTB) clinical isolates from south India. METHODOLOGY: A total of 7633 specimens from TB suspects (PTB, n = 4327 and EPTB, n = 3306) were collected during the study period (July 2018-March 2020) in a tertiary care hospital. The study specimens were subjected to Ziehl Neelsen (ZN) staining and Auramine phenol (AP) staining followed by Lowenstein-Jensen (LJ) and mycobacteria growth indicator tube (MGIT) culture. The MPT64 immunochromatographic test (ICT) was performed among mycobacterial cultures and ICT negative isolates were subjected to Line Probe Assay (LPA). In addition, 53 (PTB, 48 and EPTB, 5) NTM MGIT positive cultures were collected from Intermediate Reference Laboratory (IRL), Puducherry and subjected to LPA for speciation. RESULTS: Of the 7633 TB suspects, 0.6% were diagnosed as NTM diseases and 5.5% with Mycobacterium tuberculosis (MTBC). NTM infection was observed among 0.7% (31/4327) of PTB and 0.4% (14/3306) of EPTB. MTBC was detected among 6.1% (264/4327) of PTB and 4.6% (153/3306) of EPTB. Among 98 NTM cultures, 80.6% of isolates were recovered from PTB and 19.4% from EPTB specimens. Among pulmonary specimens, Mycobacterium intracellulare (26.6%), Mycobacterium abscessus (17.7%) and Mycobacterium kansasii (12.7%) were the most frequently detected species, while Mycobacterium intracellulare (21.1%), Mycobacterium scrofulaceum (15.8%) and Mycobacterium fortuitum (10.5%) were common in extrapulmonary specimens. CONCLUSION: The frequency of NTM infection among TB suspects was low at a South Indian tertiary care hospital. The most predominant NTM species isolated from both pulmonary and extrapulmonary specimens was M. intracellulare.

Uncommon isolation of Desulfovibrio vulgaris from a depressed fracture wound on the forehead.

Desulfovibrio spp. are gram negative, obligate anaerobes capable of reducing sulfate. They have caused infections in humans, but very rarely. They are slow growers and difficult to identify. Hence, they are often overlooked and their actual presence goes unnoticed. Here, we describe a case of a 15- year old boy who was involved in a road traffic accident and he presented with seropurulent discharge from a depressed fracture wound on the forehead. Desulfovibrio vulgaris (D.vulgaris), was isolated from the pus discharge, the first to be reported. The characteristic desulfoviridin pigment production in the organism aided in the identification. The infection was successfully managed with pain reliever and course of amoxicillin - clavulanic acid and linezolid.

Comparision of the various routine diagnostic modalities of malaria and a new method: the Parasight™ platform.

Malaria is one of most important parasitic disease, which is still much prevalent in India. The burden of malaria in India is complex and the proportions of Plasmodium vivax and Plasmodium falciparum vary across India, because of the highly variable malaria eco-epidemiological profiles, transmission factors, and the presence of multiple Plasmodium species and Anopheles vectors. The diagnostic modalities which were being used currently, are at the risk of missing potential malaria cases, if a single test is being used for a given sample. There are some extremely sensitive and specific diagnostic methods available (e.g. PCR, LAMP), but they are expensive, complex, and not readily available in all healthcare setups. Therefore, this study aimed to compare three different types of routinely used diagnostic methods and a novel testing method, the Parasight™ platform, and compare them with the detection ability of the most accurate diagnostic method, that is, PCR. A total of 111 consecutive malaria-positive (proven positive by PCR) patients were taken and tested by the immunochromatographic test or the rapid diagnostic test (RDT), thin and thick blood smears, quantitative buffy coat (QBC). In the last year of study period, 26 PCR positive samples were also taken up for the Parasight™ platform diagnostic test, along with the other routine tests. Among 111 PCR-positive cases, 78.4% samples were positive by Giemsa-stained blood film examination, 80.2% by QBC, 87.4% by RDT. In the last year of study period, among the 26 PCR-positive malaria samples, 80.8% were positive by blood film examination, 84.6% by QBC, 96.2% by RDT and 100% by the Parasight™ platform test. A combination of tests is preferable than a single method, for better detection of Plasmodium species including automated methods. The new testing method, the Parasight™ platform, is emerging to be a very sensitive test for detection of Plasmodium spp., results of which are comparable to PCR.

A comparative study assessing the effect of haematological and biochemical parameters on the pathogenesis of malaria.

Malaria is one of the most common parasitic disease affecting mankind since millennia. The most pronounced changes related to malaria involve the blood and the blood forming system, the spleen and the liver. The abnormal haematological and biochemical parameters observed in malaria cases adversely affect the prognosis of the disease. The aim of this study was to assess the severity of malaria by observing the significant abnormalities in haematological and biochemical parameters in the malaria infected cases as compared to the healthy controls. The study population comprised of 138 individuals, of which 69 were malaria cases and 69 were apparently healthy controls. All the 138 individuals were subjected to haematological and biochemical workup, following which statistical analysis was done to observe any association of altered haematological and biochemical parameters with severity of malaria, as compared to the healthy controls. Among the 138 study population, 69 patients were malaria cases whereas the other 69 were healthy controls. Haematological investigations revealed, that the haemoglobin levels, total RBC counts and haematocrit were significantly altered in the malaria cases as compared to the healthy controls. Also the leucogram profile showed significant leucopenia and neutropenia in the malaria patients as compared to the controls. Thrombocytopenia was also seen to be more pronounced in the malaria infected. The liver enzymes and serum bilirubin levels were raised in the malaria cases more than the controls. Altered haematological and biochemical parameters are indicators of disease progression to severity. Early detection and management of these parameters, will prevent the development of complications in malaria.

Sequence analysis of pfcrt and pfmdr1 genes and its association with chloroquine resistance in Southeast Indian Plasmodium falciparum isolates.

BACKGROUND: Due to the widespread resistance of Plasmodium falciparum to chloroquine drug, artemisinin-based combination therapy (ACT) has been recommended as the first-line treatment. This study aims to evaluate the extent of chloroquine resistance in P. falciparum infection after the introduction of ACT. This study was carried out based on the mutation analysis in P. falciparum chloroquine resistant transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes. Identification of these molecular markers plays a significant role in monitoring and assessment of drug resistance as well as in designing an effective antimalarial drug policy in India. METHODS: Sixty blood samples were collected from patients infected with P. falciparum from JIPMER, Puducherry and MKCG Medical College, Odisha. Polymerase chain reaction-restriction fragment length polymorphism was performed, targeting the point mutation of K76T in pfcrt and N86Y in pfmdr1 gene. The PCR products were sequenced, genotyped and further analysed for amino acid changes in these codons. RESULTS: The frequency of pfcrt mutation at 76th position was dominant for mutant T allele with 56.7% and wild type K, 43.3%. Majority of pfmdr1 86 allele were wild type, with N (90%) and mutant, Y (10%). Additionally, we found three haplotypes for CQ resistance, SVMNT, CVIET and CVIKT in association with the pfcrt gene. However, a poorly studied SNP in pfmdr1 gene (Y184F) associated with CQ resistance showed high frequency (70%) in P. falciparum isolates. CONCLUSIONS: The point mutation K76T of pfcrt is high in P. falciparum suggesting a sustained high CQ resistance even after five years of the introduction of ACTs for antimalarial therapy. The present study suggests a strong association of CQ resistance with pfcrt T76, but not with the pfmdr1 Y86 mutation. However, sequence analysis showed that Y184F mutation on pfmdr1 gene was found to be associated with high resistance. Also, a new pfcrt haplotype 'CVIKT' associated with CQ resistance was found to be present in Indian strains of P. falciparum. The data obtained from this study helps in continuous monitoring of drug resistance in malaria and also suggests the need for careful usage of CQ in Plasmodium vivax malarial treatment.