Auramine-O Staining vs Ziehl Neelsen Staining: Advantages and Disadvantages

Background: Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is still a major public health concern around the world. Prompt detection of active tuberculosis cases helps in timely therapeutic intervention and reduces community transmission. Despite limited sensitivity, conventional microscopy is still used to diagnose pulmonary tuberculosis in high-burden nations such as India. This study, therefore, was aimed at assessing the diagnostic performance of microscopy by Ziehl Neelsen (ZN) and auramine (AO) staining in the diagnosis of pulmonary tuberculosis. Materials and methods: A prospective comparative study was done on the sputum samples of 2,395 adult patients from November 2018 to May 2020 suspected of having pulmonary tuberculosis visiting the Designated Microscopic Centre of SGT Medical College, Budhera, Gurugram. Each sample was subjected to ZN staining, and AO staining as per NTEP guidelines. Results: Out of the 2,395 samples studied, 161 (6.76%) and 224 (9.35%) were positive by ZN and AO staining methods respectively. Pauci-bacillary cases detected by AO were more than ZN staining. There were 63 more sputum samples detected by AO staining which were missed by ZN microscopy. Conclusion: When compared to conventional ZN staining, the auramine staining technique is more sensitive and takes less time to diagnose pulmonary tuberculosis

Distribution of Chlamydia trachomatis omp A genotypes in patients attending a sexually transmitted disease outpatient clinic in New Delhi, India

Background & objectives: Limited data are available on the typing of Chlamydia trachomatis in India. Serovars D to K of C. trachomatis are chiefly responsible for urogenital infections. Thus, this study was conducted to determine the distribution of C. trachomatis serovars in patients with urogenital infections and to characterize omp A gene of the detected C. trachomatis isolates by sequence analysis. Presence of other co-infections was also evaluated. Methods: Endocervical swabs were collected from 324 women and urethral swabs/urine were collected from 193 men attending the sexually transmitted diseases outpatient clinic. The samples were screened for C. trachomatis by cryptic plasmid PCR and omp A gene PCR. Genotyping was performed by PCR-restriction fragment length polymorphism (RFLP) and sequencing of the omp A gene. Samples were screened for genital mycoplasmas, Neisseria gonorrhoeae, Treponema pallidum and human immunodeficiency virus (HIV). Results: C. trachomatis was found in 15.0 per cent men and 10.8 per cent women. Serovar D was the most prevalent followed by serovars E, F, I and G. Twenty two C. trachomatis isolates were selected for omp A gene sequencing. No mixed infection was found. Variability in omp A sequences was seen in 31.8 per cent cases. Both PCR-RFLP and omp A gene sequencing showed concordant results. The presence of Ureaplasma spp. and Mycoplasma hominis was observed in 18.7 and 9.5 per cent patients, respectively. Co-infection of C. trachomatis was significantly associated with Ureaplasma urealyticum and HIV. Interpretation & conclusions: The high occurence of C. trachomatis infections warrants its screening in addition to other sexually transmitted infections namely U. urealyticum and HIV. Genotyping of the omp A gene may provide additional information for vaccine development.

Use of TaqMan Array card for the detection of respiratory viral pathogens in children under 5 years old hospitalised with acute medical illness in Ballabgarh, Haryana, India

Historical specimens collected from hospitalized children were tested for the following 13 viruses: influenza A and B; respiratory syncytial virus (RSV); parainfluenza viruses 1� human metapneumovirus; rhinovirus; coronaviruses 229E, OC43, NL63 and HKU1 and Adenovirus using monoplex real-time reverse transcriptase polymerase chain reaction (rRT-PCR). They were retested using TaqMan Array Card (TAC), a micro-fluidic system, capable of simultaneous multi-pathogen testing, to evaluate its sensitivity and specificity against monoplex rRT-PCR. TAC showed high sensitivity (71%�0%) and specificity (98%�0%) for these viruses in comparison to monoplex rRT-PCR. Multi-specimen detection with high sensitivity and specificity makes TAC a potentially useful tool for both surveillance and outbreak investigations.

Respiratory syncytial virus infections in India: Epidemiology and need for vaccine

Respiratory syncytial virus (RSV) has been identified as a leading cause of lower respiratory tract infections in young children and elderly. It is an enveloped negative-sense RNA virus belonging to Genus Orthopneumovirus. The clinical features of RSV infection range from mild upper-respiratory-tract illnesses or otitis media to severe lower-respiratory-tract illnesses. Current estimates show that about 33.1 million episodes of RSV-acute lower respiratory infection (ALRI) occurred in young children in 2015, of these majority that is, about 30 million RSV-ALRI episodes occurred in low-middle-income countries. In India, the rates of RSV detection in various hospital- and community-based studies mostly done in children vary from 5% to 54% and from 8% to 15%, respectively. Globally, RSV epidemics start in the South moving to the North. In India, RSV mainly peaks in winter in North India and some correlation with low temperature has been observed. Different genotypes of Group A (GA2, GA5, NA1 and ON1) and Group B (GB2, SAB4 and BA) have been described from India. The burden of RSV globally has kept it a high priority for vaccine development. After nearly 50 years of attempts, there is still no licensed vaccine and challenges to obtain a safe and effective vaccine is still facing the scientific community. The data in this review have been extracted from PubMed using the keywords RSV and Epidemiology and India. The data have been synthesised by the authors.

Molecular characterisation of Staphylococcus aureus using spa typing as a diagnostic tool in Haryana, India

Background: Staphylococcus aureus is one of the top six most common etiologic agents of nosocomial, community and livestock acquired bacterial infections. These infections although initially were described as a major problem in hospitals have now also become a serious threat in community not only in India but also worldwide. Its prevalence varies depending on the health-care setting, country or a particular region. Thus to better understand the epidemiology of methicillin-resistant S. aureus (MRSA) in a particular geographical location, it is important to study the variations in the population using molecular tools. Methods: This prospective study was carried out in the Department of Microbiology of Shree Guru Gobind Singh Tricentenary (SGT) Medical College. Staphylococcal protein A (spa) typing was done on 250 S. aureus isolates obtained from various clinical specimens including pus, wound swabs, urine, catheters, blood and cerebrosspinal fluid from both indoor and outdoor patients of SGT Hospital, Budhera, Gurgaon. Results: The selected region of the spa gene of all 250 isolates which includes 87 MRSA and 163 methicillin-susceptible S. aureus were amplified. The spa gene was detected in 248 out of 250 isolates (99.2%), whereas in 2 isolates (0.8%), it remained undetected and referred as non-typable isolates. The 248 S. aureus isolates were typed into 39 spa types, which clustered into six different spa clonal clusters and eight singletons. Conclusion: High diversity observed within S. aureus isolates indicated that many different strains circulate in the study region or in the hospital. The results would contribute in the understanding of epidemiology related to S. aureus spread and prevention.

Comparison of multiplex RT-PCR with virus isolation for detection, typing and sub-typing of influenza virus from influenza-like illness cases.

Purpose: Infl uenza epidemics and periodic pandemics occur worldwide resulting in signifi cant mortality, morbidity and economic loss. There is need for a sensitive, rapid and cost-effective assay to detect, type and sub-type infl uenza viruses, as cell culture has a long turnaround time. Materials and Methods: Nasopharyngeal swabs were collected from patients presenting with infl uenza-like illness (ILI) at AIIMS OPD and Primary Health Centre Ballabhgarh (Haryana). From June 2007 to January 2009 and then from September to November 2009, of 1567 specimens collected, 544 were randomly selected and were tested by virus culture using Madin-Darby Canine Kidney (MDCK) cells and by reverse transcription polymerase chain reaction (RT-PCR) for infl uenza A using primers for matrix gene and for infl uenza B using non-structural gene (NS) primers. All infl uenza A positives were sub-typed using primers for HA and NA genes of A/H1, A/H3. A separate multiplex RT-PCR having primers from matrix and HA genes of pandemic A (H1N1) pdm09 viruses was carried out on samples collected after September 2009. Results: Of the 544 samples, 136 (25%) were positive for infl uenza by RT-PCR. Further typing analysis revealed 86 (63.2%) were typed as infl uenza A and 47 (34.5%) as infl uenza B viruses and 3 (2%) samples showed dual infection with infl uenza A and B. Of the 86 infl uenza A positive samples 48 (55.8%) were identifi ed as seasonal infl uenza A/H1N1, 22 (25.6%) as A (H1N1) pdm09 and 16 (18.6%) as A/H3N2. Comparison of infl uenza positivity using virus culture revealed that only 97/136 (71.3%) were infl uenza positive. Sensitivity of viral detection was lowest for seasonal A/H1 (26/48; 54%), followed by H3N2 (11/16; 68.7%) and infl uenza B (38/47; 80.8%); all infl uenza A/H1N1pdm09 viruses were detected by both methods. Conclusion: RT-PCR is a sensitive, low cost and rapid screening test for diagnosing infl uenza infection during epidemics and pandemics. mRT-PCR increased the detection rates for infl uenza by 28.6% as compared with virus isolation and thus is a useful assay in both diagnostic and epidemiological settings in resource poor countries.

Antiviral drug profile of human influenza A & B viruses circulating in India: 2004-2011.

background & objectives: Recent influenza antiviral resistance studies in South East Asia, Europe and the United States reveal adamantane and neuraminidase inhibitor (NAIs) resistance. This study was undertaken to evaluate antiviral resistance in influenza viruses isolated from various parts of India, during 2004 to 2011. methods: Influenza viruses were analyzed genetically for known resistance markers by M2 and NA gene sequencing. Influenza A/H1N1 (n=206), A/H3N2 (n=371) viruses for amantadine resistance and A/H1N1 (n=206), A/H3N2 (n=272) and type B (n=326) for oseltamivir resistance were sequenced. Pandemic (H1N1) (n= 493) isolates were tested for H274Y mutation by real time reverse transcription (rRT)-PCR. Randomly selected resistant and sensitive influenza A/H1N1 and A/H3N2 viruses were confirmed by phenotypic assay. results: Serine to asparagine (S3IN) mutation was detected in six isolates of 2007-2008.One dual-resistant A/H1N1 was detected for the first time in India with leucine to phenylalanine (L26F) mutation in Mm2 gene and H274Y mutation in NA gene. A/H3N2 viruses showed an increase in resistance to amantadine from 22.5 per cent in 2005 to 100 per cent in 2008 onwards with S3IN mutation. Fifty of the 61 (82%) A/H1N1 viruses tested in 2008-2009 were oseltamivir resistant with H274Y mutation, while all A/H3N2, pandemic A/H1N1 and type B isolates remained sensitive. Genetic results were also confirmed by phenotypic analysis of randomly selected 50 resistant A/H1N1 and 40 sensitive A/H3N2 isolates. Interpretation & conclusions: Emergence of influenza viruses resistant to amantadine and oseltamivir in spite of negligible usage of antivirals emphasizes the need for continuous monitoring of antiviral resistance.

Seroepidemiological study of human metapneumovirus in New Delhi, India.

Purpose: There are a few seroepidemiological studies reported on human metapneumovirus (hMPV) as hMPV was only discovered in the year 2001. This respiratory virus has been reported to be ubiquitous and associated with respiratory tract infections in all age groups. The present study aimed at determining the prevalence of antibodies to hMPV in children and adults of 1 month to 55 years of age. Materials and Methods: Serum samples from 100 study subjects were tested for hMPV antibody by an in-house ELISA system that used hMPV-infected cell lysate antigen. Result: The prevalence of antibody to hMPV was lowest in children less than 5 years of age (60%) and increased throughout age to > 80%. Similarly, geometric mean titres were 1:180 in children less than 5 years of age and reached a peak of 1:419 in adults over 35 years of age. Conclusion: The results show that hMPV infection is acquired early in life and re-infection in later life may maintain the seroprevalence and antibody levels in adult population.

Epidemiology and symptom profile of gastroesophageal reflux in the Indian population: Report of the Indian Society of Gastroenterology Task Force.

Background Gastroesophageal reflux disease (GERD) and its complications are thought to be infrequent in India; there are no data from India on the prevalence of and risk factors for GERD. The Indian Society of Gastroenterology formed a task force aiming to study: (a) the frequency and profile of GERD in India, (b) factors including diet associated with GERD. Methods In this prospective, multi-center (12 centers) study, data were obtained using a questionnaire from 3224 subjects regarding the frequency, severity and duration of heartburn, regurgitation and other symptoms of GERD. Data were also obtained regarding their dietary habits, addictions, and lifestyle, and whether any of these were related or had been altered because of symptoms. Data were analyzed using univariate and multivariate methods. Results Two hundred and forty-five (7.6%) of 3224 subjects had heartburn and/or regurgitation at least once a week. On univariate analysis, older age (OR 1.012; 95% CI 1.003–1.021), consumption of non-vegetarian and fried foods, aerated drinks, tea/coffee were associated with GERD. Frequency of smoking was similar among subjects with or without GERD. Body mass index (BMI) was similar in subjects with and without GERD. On multivariate analysis, consumption of non-vegetarian food was independently associated with GERD symptoms. Overlap with symptoms of irritable bowel syndrome was not uncommon; 21% reported difficulty in passage of stool and 9% had mucus in stools. About 25% of patients had consulted a doctor previously for their gastrointestinal symptoms. Conclusion 7.6% of Indian subjects have significant GERD symptoms. Consumption of non-vegetarian foods was an independent predictor of GERD. BMI was comparable among subjects with or without GERD.

Clinical Profile and Outcome of Swine Flu in Indian Children.

Objective: To describe the clinical characteristics and outcome of Indian children infected with 2009 H1N1 influenza virus. Study design: Retrospective chart review. Setting: Outpatient department and hospitalized patients in a tertiary care hospital. Methods: Clinical details of 85 children (positive for the 2009 H1N1 virus infection tested by real-time reversetranscriptase– polymerase-chain-reaction assay) were analyzed from medical charts. Results: Of the 85 (55 boys) children positive for 2009 H1N1 virus infection, 64.7% were between 5 years to 16 years, and 35.3% were below 5 years age. The mean age of these children was 7.5±3.5 yr. Contact history was positive only in 22 (26%) cases. High grade fever was the most common symptom, followed by cough and rhinorrhea. Twenty-nine (34%) patients had an underlying co-morbid condition. Of the 34 patients who underwent chest radiography during evaluation, 18 children (52.9%) had findings consistent with lower respiratory tract infection. Antiviral therapy was initiated in 76 patients. Hospitalization was required in 30 (35.3%) children. Risk factors for hospitalization included underlying co-morbid condition, respiratory distress, vomiting, wheezing, diarrhea, hypotension and infiltrates/consolidation on chest radiograph. Mean length of hospitalization was 131+76 hours, irrespective of underlying disease. Three children developed Acute Respiratory Distress Syndrome and died. Conclusions: Clinical features and routine laboratory investigations in children with swine origin influenza were non-specific. Children with co-morbid condition, respiratory distress, vomiting, wheezing, diarrhea, hypotension and infiltrates/consolidation on chest radiograph were at higher risk of hospitalization.

Human metapneumovirus: a new respiratory pathogen.

Human metapneumovirus is a recently recognized pathogen of acute respiratory tract infection (ARI) in children as well as elderly and immunocompromised adults.The virus belongs to the family Paramyxoviridae, sub family Pneumovirinae and genus Metapneumovirus. Through genetic analysis it has been characterized into two groups A and B which are further divided into four sub-lineages. The virus is difficult to grow in tissue culture and hence reverse transcriptase-polymerase chain reaction (RT-PCR) for N and L gene is the method of choice for diagnosis. The virus has been seen in all countries with seasonal distribution in winter months for temperate and spring/summer for tropical countries. F gene is the most conserved among different lineages and efforts are underway to design recombination vaccine using F gene.

Duodenal angiomyolipoma: a case report.

The authors present a case of duodenal angiomyolipoma. Angiomyolipoma is a benign neoplasm, the most common site being the kidney. Extrarenal angiomyolipomas are rare. Only one case of duodenal angiomyolipoma has been reported. A 66 year old man presented with GI bleeding for two years. Endoscopy revealed a duodenal polyp which on histopathology revealed features of angiomyolipoma. On immunohistochemistry, smooth muscle component was positive for Smooth Muscle Actin. HMB-45 showed mild focal positivity. Patient had no history of tuberous sclerosis. Surgical excision of angiomyolipoma is usually curative with rare cases of local recurrence.

Role of CSF serology in follow-up of subacute sclerosing panencephalitis patients on treatment.

Subacute sclerosing panencephalitis (SSPE) is a progressive inflammatory disease of the central nervous system with poor prognosis and high mortality. No effective treatment has a proven role; oral isoprinosine and intrathecal administration of alpha-interferon may prolong survival. We report an unusual case of adult onset SSPE patient on treatment with significant clinical improvement, even in the absence of conversion to seronegativity in either CSF or serum, on follow-up serological examination.

Can we identify acute severe viral lower respiratory tract infection clinically?

Two hundred children below five years of age hospitalized with a clinical diagnosis of acute severe lower respiratory tract infection were enrolled in the study. Nasopharyngeal (NP) aspirate was collected for viral isolation by centrifugation enhanced culture technique. Viruses were isolated from 89 NP aspirates. Clinical features of these 89 children were compared with 111 children whose NP aspirates were negative for viruses. There was significantly higher incidence of breathlessness and rhonchi in children whose nasopharyngeal aspirates yielded virus. Sensitivity,specificity, positive and negative predictive values of breathlessness for severe viral ALRTI were 98%, 10.8%, 46.8% and 85%, respectively. The values for rhonchi were 60%, 56.8%, 58.2%, and 74.1%, respectively. It is concluded that clinical features do not have desirable sensitivity and specificity for identification of ALRTI due to viral etiology.

Expression and humoral immune response to hepatitis C virus using a plasmid DNA construct.

PURPOSE: The objective of this study was to clone a c-DNA fragment of hepatitis C virus in a eukaryotic expression vector and to measure the efficacy of humoral immune responses in mice inoculated with this recombinant plasmid. This study was an attempt to lay a foundation for HCV nucleic acid vaccine development in the future. METHODS: A c-DNA fragment of BK146, a clone of HCV type 1b, was sub-cloned into an eukaryotic expression vector pMT3. HepG2 and COS cells were transfected with this construct, named pMT3-BK146. The expression of HCV mRNA and proteins was studied by reverse transcribed polymerase chain reaction, radio Immunoprecipitation (RIPA) and immunofluorescence (IFA). The DNA of this construct was injected into the footpad of BALB/c mice and antibody response was tested by enzyme immunoassay and indirect immunofluorescence. RESULTS: COS and HepG2 cells transiently transfected with the recombinant plasmid pMT3-BK146 showed the expression of HCV proteins by RT-PCR, RIPA and immunofluorescence. This DNA clone when injected into Balb/c mice was able to generate specific antibody response to hepatitis C virus by ELISA and IFA. CONCLUSIONS: A c-DNA fragment of HCV cloned in an eukaryotic expression vector was able to express core protein. This DNA clone was also able to elicit antibody response in mice. This can be an initial step towards the development of a potential DNA vaccine for hepatitis C virus infection.

Etiology of acute lower respiratory tract infection.

OBJECTIVE: To identify pathogens responsible for acute severe lower respiratory tract infection (ALRTI) in under five children by non-invasive methods. METHOD: 95 children hospitalized with acute severe lower respiratory tract infection were investigated for identification of viruses, bacteria, chlamydia or mycoplasma by nasopharyngeal aspirates, blood culture and serology. RESULT: Etiological agents could be identified in 94% of the patients. Viruses from NP aspirate could be isolated in 36 (38%), bacterial isolates from blood cultures in 15 (16%); mycoplasma was identified in 23 (24%) and chlamydia in 10 (11%) by serological tests; mixed infections were present in 8 (8%) patients. CONCLUSION: Noninvasive methods can be useful in identifying etiological agents in severe ALRTI.

Risk factors for severe acute lower respiratory tract infection in under-five children.

BACKGROUND: Acute lower respiratory infection (ALRTI) is the leading cause of death in children below five years of age. Identification of modifiable risk factors of severe ALRTI may help in reducing the burden of disease. METHODS: A hospital based case control study was undertaken to determine risk factors associated with severe lower respiratory tract infection (LRTI) in under-five children. A case definition of severe ALRTI as given by World Health Organization (WHO) was used for cases. Healthy children attending Pediatrics out patient department for immunization during study period were enrolled as controls. Details of potential risk factors in cases and controls were recorded in pre-designed proforma. RESULTS: 512 children including 201 cases and 311 controls were enrolled in the study. On stepwise logistic regression analysis it was found that lack of breastfeeding (OR: 1.64; 95 percent CI: 1.23-2.17); upper respiratory infection in mother (OR: 6.53; 95 percent CI: 2.73-15.63); upper respiratory infection in siblings (OR: 24; 95 percent CI: 7.8-74.4); severe malnutrition (OR: 1.85; 95 percent CI: 1.14-3.0); cooking fuel other than liquid petroleum gas (OR: 2.5; 95 percent CI: 1.51-4.16); inappropriate immunization for age (OR: 2.85; 95 percent CI 1.59-5.0) and history of LRTI in the family (OR 5.15, 95 percent CI 3.0-8.8) were the significant contributors of ALRTI in children under five years. Sex of the child, age of the parents, education of the parents, number of children at home, anemia, inadequate caloric intake, type of housing were not documented to be significant risk factors of ALRTI. CONCLUSION: Lack of breast-feeding, upper respiratory infection in mother, upper respiratory infection in siblings, severe malnutrition, cooking fuel other than liquid petroleum gas, inappropriate immunization for age and history of LRTI in the family were the significant risk factors associated with ALRTI

Comparative evaluation of various commercial assays for diagnosis of dengue fever.

Dengue fever (DF) is endemic in India and dengue hemorrhagic fever (DHF) has been reported with increasing frequency in the last decade. We evaluated three commercial assays for detection of antibodies to dengue virus, to assess their performance in a diagnostic laboratory. Sera from 58 patients collected during a febrile outbreak in New Delhi in 1997 were studied. The methods evaluated were MRL Diagnostic Dengue Fever Virus IgM Capture ELISA, Pan Bio Dengue Duo IgM and IgG Capture ELISA and Pan Bio Rapid Immunochromatographic test. The MRL ELISA correctly identified 97.8% (43 of 44) of samples as dengue positive while the Pan Bio Duo ELISA and Pan Bio RIT identified 95.45% (42 of 44). The sensitivities of both Pan Bio Duo ELISA and Pan Bio RIT for primary dengue and secondary dengue were 100% and 93.54% respectively. The specificity of three assays were MRL IgM ELISA 100%, Pan Bio Duo ELISA 92.8% and Pan Bio RIT 85.7%.