Bacteriological profile and antibiotic sensitivity pattern of neonatal septicaemia in a rural tertiary care hospital in North India

Background: There is not much published literature on neonatal septicemia available for the Sub-Himalayan region of North India. Hence, we undertook this study to find out the bacteriological profile and antibiotic sensitivity pattern of neonatal septicemia in the neonatal Intensive Care Unit. Material and Methods: Blood cultures were performed for all clinically suspected neonatal septicemia cases for 1-year. Identification of all pathogenic isolates was followed by antibiotic sensitivity testing. Results: We did blood cultures for 450 neonates and 42% were culture positive. Early onset sepsis were 92 (49%) and 96 (51%) were late onset sepsis. Gram-positive isolates were 60% and 40% were Gram-negative. Staphylococcus aureus (40%), coagulase negative Staphylococcus species (16%), non-fermenter group of organisms (NFGOs) (15%), and Klebsiella pneumoniae (10%) were the main isolates. Nasal cannula 101 (54%), birth asphyxia 91 (48%), and prematurity 73 (38%) were the prominent risk factors associated with septicemia. Gram-positive organisms were highly resistant to penicillin (87%) whereas Gram-negative isolates showed high resistance to third generation cephalosporins (53–89%) and aminoglycosides (50–67%). The S. aureus isolates were methicillin-resistant in 41% whereas extended spectrum beta lactamase production was seen in 48% Gram-negative isolates. Conclusion: Our study highlights the recent emergence of Gram-positive organisms as predominant cause of neonatal septicemia in this part of Sub-Himalayan region, along with the review of literature which shows similar results from North India and rest of the world too. Though Gram-negative bacteria still remain the main cause of mortality in neonatal septicemia, we want to dispel the common notion among practitioners that they are the predominant isolates in neonatal septicemia.     

Predictors of the extended-spectrum-beta lactamases producing Enterobacteriaceae neonatal sepsis at a tertiary hospital,Tanzania

The study was conducted to establish predictors of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) neonatal sepsis and mortality in a tertiary hospital, Tanzania. Between July and December 2016, blood culture was performed in neonates with clinical features of sepsis and neonates/mothers/guardians were screened for ESBL colonization. Selected isolates underwent whole genome sequencing to investigate relatedness. Logistic regression analysis was performed to determine predictors for ESBL-PE associated neonatal sepsis and mortality. Neonatal ESBL-PE sepsis was detected in 32(10.5%) of the 304 neonates investigated. Neonatal ESBL-PE sepsis was independently predicted by admission at the Intensive care Unit and positive mother and neonate ESBL-PE colonization. Deaths occurred in 55(18.1%) of neonates. Neonates infected with ESBL-PE, admitted at ICU, increased age and those transferred from other centres had significantly high mortality rates. Gram-negative bacteria formed the majority (76%) of the isolates, of which 77% were ESBL-PE. Virulent Klebsiella pneumoniae ST45 carrying bla_CTX-M-15 were commonly isolated from neonates. Klebsiella pneumoniae (ST45) were the predominant cause of ESBL-PE neonatal sepsis and mortality. Improved infection control and antibiotic stewardship are crucial in controlling the spread of resistant strains. Rapid diagnostic tests to detect ESBL-PE in low-income countries are needed to guide treatment and reduce ESBL-PE-associated mortality.     

Molecular Confirmation of the Circulating Bacillus anthracis during Outbreak of Anthrax in Different Villages of Simdega District,Jharkhand

Aims and Objectives: Molecular confirmation of the circulating Bacillus anthracis during outbreak of anthrax in different villages of Simdega district, Jharkhand, India. Materials and Methods: Blood samples with swabs from skin lesions (eschar) were collected from the suspected cases of Anthrax from October 2014 to June 2016 from Simdega district, Jharkhand. All the swabs were inoculated on polymyxin lysozyme EDTA thallous acetate media, nutrient agar media as well as 5% sheep blood agar media. Gamma-phage lysis was done. DNA extraction was done using a QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA) and subjected to polymerase chain reaction (PCR) using anthrax-specific primers. Results: On Gram and acid fast staining, purple rods and pink-coloured anthrax spores were detected. Capsular and M’Fadyean staining was done. Gamma-phage lysed B. anthracis culture. Of 39 suspected cases, 8 were culture and PCR positive and showed gamma-phage lysis. 3 deaths were reported. Discussion and Conclusion: The conventional and real-time PCR methods are suitable for both the clinical and the epidemiological practice.     

Blood culture gram stain,acridine orange stain and direct sensitivity-based antimicrobial therapy of bloodstream infection in patients with trauma

Purpose: The purpose of this study was to ascertain if the simple practice of Gram stain, acridine orange stain and direct sensitivity determination of positive blood culture bottles could be used to guide early and appropriate treatment in trauma patients with clinical suspicion of sepsis. The study also aimed to evaluate the error in interpreting antimicrobial sensitivity by direct method when compared to standard method and find out if specific antibiotic–organism combination had more discrepancies. Findings from consecutive episodes of blood stream infection at an Apex Trauma centre over a 12-month period are summarized. Materials and Methods: A total of 509 consecutive positive blood cultures were subjected to Gram staining. AO staining was done in BacT/ALERT-positive Gram-stain negative blood cultures. Direct sensitivity was performed from 369 blood culture broths, showing single type of growth in Gram and acridine orange staining. Results of direct sensitivity were compared to conventional sensitivity for errors. Results: No ‘very major’ discrepancy was found in this study. About 5.2 and 1.8% minor error rates were noted in gram-positive and gram-negative bacteria, respectively, while comparing the two methods. Most of the discrepancies in gram-negative bacteria were noted in β lactam – β lactamase inhibitor combinations. Direct sensitivity testing was not reliable for reporting of methicillin and vancomycin resistance in Staphylococci. Conclusions: Gram stain result together with direct sensitivity testing is required for optimizing initial antimicrobial therapy in trauma patients with clinical suspicion of sepsis. Gram staining and AO staining proved particularly helpful in the early detection of candidaemia.     

Detection of 123 bp fragment of insertion element IS6110 Mycobacterium tuberculosis for diagnosis of extrapulmonary tuberculosis

Purpose: Extrapulmonary tuberculosis (EPTB) is emerging problem in developing and developed countries. The diagnosis of EPTB in its different clinical presentations remains a true challenge. IS6110-based polymerase chain reaction (PCR) is used for rapid identification and positivity rate of the Mycobacterium tuberculosis complex in clinical isolates of different sites of EPTB. The present study was carried out to study the prevalence of M. tuberculosis complex in clinical isolates of EPTB at tertiary care centres in Lucknow. Materials and Methods: Seven hundred fifty-six specimens were collected from the suspected cases of EPTB which were processed for Mycobacteria by Ziehl Neelson (ZN) staining and BACTEC culture. All the specimens were also processed for IS6110-based PCR amplification with primers targeting 123 bp fragment of insertion element IS6110 of the M. tuberculosis complex. Results: Of these 756 specimens, 71(9.3%) were positive for acid fast bacilli (AFB) by ZN staining, 227(30.1%) were positive for mycobacteria by BACTEC culture and IS6110 PCR were positive for M. tuberculosis complex in 165 (20.7%) isolates. We found a significant difference in sensitivities of different tests (P<0.05). Conclusions: This study reveals the positivity of M. tuberculosis complex in clinical isolates of EPTB case in tertiary care hospitals in Northern India. 72.7% of M. tuberculosis complex was confirmed by IS6110-PCR in culture isolates from different sites of EPTB. The high prevalence of the M. tuberculosis complex was seen in lymph node aspirate and synovial fluid. However, utility of PCR may play a potentially significant role in strengthening the diagnosis of EPTB especially targeting IS6110.     

An Outbreak of Burkholderia cepacia Complex in the Paediatric Unit of a Tertiary Care Hospital

Introduction: Burkholderia cepacia complex (Bcc) has emerged as a serious nosocomial pathogen worldwide especially in patients with indwelling catheters and cystic fibrosis. Bcc is a common contaminant of pharmaceutical products. We describe an outbreak of Bcc bacteraemia amongst children admitted in Paediatric Intensive Care Unit (PICU) and paediatric ward at a tertiary care hospital, Mumbai, in Western India. Materials and Methods: Blood culture samples from paediatric patients yielded growth of non-fermenting, oxidase positive, motile, Gram negative bacilli (NFGNB) (76/909) over a period of 8 months. Based on conventional biochemical tests and antimicrobial susceptibility testing, these isolates were provisionally identified as Bcc. The increased, repeated and continued isolation of Bcc alerted the possibility of an outbreak confined to PICU and paediatric ward. Active surveillance was undertaken to trace the source and contain the outbreak. Isolates were subjected to recA polymerase chain reaction (PCR) and Expanded multilocus sequence typing (EMLST). Results: Surveillance revealed the presence of Bcc on the upper surface of rubber stopper of sealed multidose amikacin vials. Isolates from blood culture and rubber stoppers were confirmed as Bcc by recA PCR. EMLST revealed that these isolates shared an identical novel sequence type 824 proving clonality. Timely interventions instituted led to control of the outbreak. Conclusion: This study highlights the importance of identification and molecular characterization of Bcc to establish its role in infection and outbreak.     

Bacteriological profile of community acquired acute bacterial meningitis: a ten-year retrospective study in a tertiary neurocare centre in South India

Purpose: Ten years retrospective study to evaluate the bacteriological spectrum of community acquired acute bacterial meningitis (CAABM). Methods: Cerebrospinal fluid (CSF) samples from 385 clinically suspected cases of pyogenic meningitis were processed for cell counts, cytospin Gram stain, culture, antigen detection by latex agglutination (LAT) and antibiotic susceptibility test. Eighteen of these CSF samples were also subjected to a polymerase chain reaction (PCR) assay for detection of pneumococcal DNA. Results: The etiological agent could be identified in 284 (73.8%) of the total 385 cases by culture and/or smear and /or LAT. Streptococcus pneumoniae was the predominant pathogen accounting for 238 (61.8%) cases. Haemophilus influenzae and Neisseria meningitidis accounted for 7 (1.8%) and 4 (1%) cases respectively. Other gram negative bacilli, Streptococcus spp. and Staphylococcus aureus were isolated from 19 (4.9%), 9 (2.3%) and 7 (1.8%) cases respectively. Conclusions: Streptococcus pneumoniae remains the major aetiological agent of CAABM both in adults and children in our set-up. No penicillin resistance was detected among the isolates. Further research should focus on preventable aspects of CAABM, especially pneumococcal vaccines, to help reduce the disease burden.     

Diagnostic potential of IS6110, 38kDa, 65kDa and 85B sequence-based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical samples

Purpose: The correlation between the presence of specific gene sequence of M. tuberculosis and specific diagnosis of clinical tuberculosis is not known. This study compared the results of polymerase chain reaction (PCR) amplification of M. tuberculosis specific DNA sequences (IS6110, 65kDa, 38kDa and mRNA coding for 85 B protein) from different clinical samples of pulmonary and extrapulmonary tuberculosis. Methods: One hundred and seventy-two clinical samples from suspected tuberculosis patients were tested for smear examination, culture (LJ and rapid BACTEC 460 TB system) and PCR. PCR was performed with specific primers for the targets: IS6110, 65kDa, 38kDa and 85B. Results: Each PCR test was found to have a much higher positivity than conventional test and BACTEC culture (P 0.05). Smear positive samples (56) and the samples (36) showing positive results by conventional methods (smear and LJ medium culture) and BACTEC were found to be positive by all PCR protocols. No significant difference was found between the four PCR protocols (P >0.05). The primer specific for amplifying the 123bp IS6110 fragment gave the highest positivity (83%), followed by 65kDa, 38kDa and 85B RT-PCR in descending order. Conclusions: These data suggest that the presence of IS6110 correlates more closely with the diagnosis of clinical tuberculosis than that of 65kDa, 38kDa and 85B     

Chlamydia trachomatis causing neonatal conjunctivitis in a tertiary care center

Chlamydia trachomatis is considered a major aetiological agent of conjunctivitis in newborns. The objective of the present study was to determine the aetiology of neonatal conjunctivitis and clinico-epidemiological correlates of chlamydial ophthalmia neonatorum. Fifty-eight newborns with signs and symptoms of conjunctivitis were studied. Conjunctival specimens were subjected to Gram staining, routine bacteriological culture, culture for Neisseria gonorrhoeae and direct fluorescent antibody (DFA) staining for diagnosis of C. trachomatis infection. C. trachomatis was detected in 18 (31%) neonates. Findings suggest that since C. trachomatis is the most common cause of neonatal conjunctivitis, routine screening and treatment of genital C. trachomatis infection in pregnant women and early diagnosis and treatment of neonatal Chlamydial conjunctivitis may be considered for its prevention and control.     

Molecular analysis of Rv0679c and Rv0180c genes of Mycobacterium tuberculosis from clinical isolates of pulmonary tuberculosis

Context: Two novel proteins/genes Rv0679c and Rv0180c of Mycobacterium tuberculosis (MTB) H37Rv were classified as a hypothetical membrane and transmembrane proteins which might have a role in the invasion. Molecular analysis of these genes in human clinical isolates of pulmonary tuberculosis (PTB) patients was not well characterised. Aims: To assess the molecular diversity of Rv0679c and Rv0180c genes of MTB from clinical isolates of PTB patients. Settings and Design: DNA from 97 clinical isolates was extracted and subjected to amplification using selective primers by polymerase chain reaction (PCR). The PCR product obtained was sequenced commercially. Patients and Methods: Clinical isolates obtained from tuberculosis patients were investigated for polymorphisms in the Rv0679c and Rv0180c genes by PCR and DNA sequencing. Genomic DNA isolated by cetyltrimethylammonium bromide method was used for amplification of genes. Results: Rv0679c gene was highly conserved in 61 out of 65 clinical isolates assessed for sequence homology with wild-type H37Rv gene and was identical using ClustalW. Fifty-five out of 78 (70.5%) clinical isolates assessed for Rv0180c were positive for single nucleotide polymorphism (SNP) at 258^th position where the nucleotide G was replaced with T (G to T). In clinical isolates of untreated cases, the frequency was 54.5% for SNP at 258^th position which is low compared to cases undergoing treatment where the frequency was 73.1%. Conclusions: Molecular analysis of Rv0180c in clinical isolates of PTB assessed in this study was the first report, where an SNP at 258^th position G to T was identified within the gene. Rv0679c gene was highly conserved (94%), within Indian clinical isolates as compared to reports from other nations.     

First description of SHV-148 mediated extended-spectrum cephalosporin resistance among clinical isolates of Escherichia coli from India

Purpose: The present study was aimed to investigate the genetic context, association with IS26 and horizontal transmission of SHV-148 among Escherichia coli in Tertiary Referral Hospital of India. Methodology: Phenotypic characterisation of extended-spectrum beta-lactamases (ESBLs) was carried out as per CLSI criteria. Molecular characterisation of bla_SHV and integron was carried out by polymerase chain reaction (PCR) assay and confirmed by sequencing. Linkage of IS26 with bla_SHV-148 was achieved by PCR. Purified products were cloned on pGEM-T vector and sequenced. Strain typing was performed by pulsed field gel electrophoresis with XbaI digestion. Transferability experiment and antimicrobial susceptibility was performed. Results: A total of 33 isolates showed the presence of SHV-148 variant by sequencing and all were Class 1 integron borne. PCR and sequencing results suggested that all bla_SHV-148 showed linkage with IS26 and were present in the upstream portion of the gene cassette and were also horizontally transferable through F type of Inc group. Susceptibility results suggest that tigecycline was most effective. Conclusion: The present study reports for the first time of SHV-148 mediated extended spectrum cephalosporin resistance from India. Association of their resistance gene with IS26 and Class 1 integron and carriage within IncF plasmid signifies the potential mobilising unit for the horizontal transfer.     

Application of polymerase chain reaction to differentiate herpes simplex virus 1 and 2 serotypes in culture negative intraocular aspirates

Purpose: To standardize and apply a polymerase chain reaction (PCR) on the glycoprotein D gene to differentiate Herpes simplex virus (HSV) 1 &; 2 serotypes in culture negative intraocular specimens. Methods: Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR) for DNA polymerase gene. To differentiate HSV serotypes, as 1 &; 2, a seminested PCR (snPCR) targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV) and varicella zoster virus (VZV) by nucleic acid amplification methods. Results: Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens), and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others. Conclusions: The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.     

Use of Lambda Phage DNA as a Hybrid Internal Control in a PCR-Enzyme Immunoassay To Detect Chlamydia pneumoniae

An inherent problem in the diagnostic PCR assay is the presence of ill-defined inhibitors of amplification which may cause false-negative results. Addition of an amplifiable fragment of foreign DNA in the PCR to serve as a hybrid internal control (HIC) would allow for a simple way to identify specimens containing inhibitors. Two oligonucleotide hybrid primers were synthesized to contain nucleic acid sequences of the Chlamydia pneumoniae 16S rRNA primers in a position flanking two primers that target the sequences of a 650-bp lambda phage DNA segment. By using the hybrid primers, hybrid DNA comprising a large sequence of lambda phage DNA flanked by short pieces of chlamydia DNA was subsequently generated by PCR, cloned into a plasmid vector, and purified. Plasmids containing the hybrid DNA were diluted and used as a HIC by adding them to each C. pneumoniae PCR test. Consequently, C. pneumoniae primers were able to amplify both chlamydia DNA and the HIC DNA. The production of a 689-bp HIC DNA band on an acrylamide gel indicated that the specimen contained no inhibitors and that internal conditions were compatible with PCR. Subsequently, a biotinylated RNA probe for the HIC was transcribed from a nested sequence of the HIC and was used for its hybridization. Detection of the HIC DNA-RNA hybrid was achieved by enzyme immunoassay (EIA). This PCR-EIA system with a HIC was initially tested with 12 previously PCR-positive and 14 previously PCR-negative specimens. Of the 12 PCR-positive specimens, 11 were reconfirmed as positive; 1 had a negative HIC value, indicating inhibition. Of the 14 previously PCR-negative specimens, 13 were confirmed as true negative; 1 had a negative HIC value, indicating inhibition. The assay was then used with 237 nasopharyngeal specimens from patients with pneumonia. Twenty-one of 237 (8.9%) were positive for C. pneumoniae, and 42 (17.7%) were found to inhibit the PCR. Specimens showing inhibitory activity were diluted 1:10 and were retested. Ten specimens were still inhibitory to the PCR and required further DNA purification. No additional positive samples were detected and 3 nasopharyngeal specimens remained inhibitory to PCR. Coamplification of a HIC DNA can help confirm true-negative PCR results by ruling out the presence of inhibitors of DNA amplification.     

Hepatitis B Virus X Protein: The X Factor in Chronic Hepatitis B Virus Disease Progression

Introduction: Hepatitis B virus (HBV) is the most common aetiological factor causing hepatocellular carcinoma (HCC). HBx gene plays an enigmatic role in HBV-related HCC. In this study we have analysed amino acid substitutions in HBx from HBV-infected individuals of different clinical stages. Materials and Methods: HBV-infected individuals (n = 93) were recruited in the study. DNA was extracted from plasma, amplified, and DNA sequencing was performed using specific primers targeting HBx gene (540 bp). Results: Among the study participants, 57% had chronic HBV infection, 30% had chronic liver disease (CLD) and 13% had HBV related HCC. Genotypes such as D1, D2, D3, A1, C2 and B2 were identified of which genotype D2 was predominant (78%). HBxC-terminal deletion was observed in four hepatitis B e antigen (HBeAg) negative participants with CLD. The frequency of aminoacid substitution in proapoptotic domain was higher in HBeAg negative participants including I127V (34%), K130M (34%), V131I (40%). The frequency of double mutation (K130M+V131I) and triple mutation (I127V+K130M+V131I) were found to be higher (32% and 36%) in HBeAg negative participants. Also, we identified L5M substitution (4.3%) in HBeAg positive participants with advanced liver disease. Conclusion: In HBx gene, aminoacid substitutions at positions 127, 130, 131 are associated with poor expression of HBeAg. We suggest screening for HBx aminoacid substitutions especially in patients with HBeAg negative chronic HBV infection to predict the clinical outcome and enable early treatment to prevent disease progression.     

Standardization of fungal polymerase chain reaction for the early diagnosis of invasive fungal infection

Background: An early initiation of antifungal therapy in invasive fungal infections (IFIs) is critical in reducing the high mortality rate. Current diagnosis of fungal infection relies on microscopy, culture, antigen, antibody specific tests and histological diagnosis. However, these tests either lack sensitivity or specificity. There is thus the need for a rapid, specific and accurate diagnostic method. Objective: The aim of our study was to establish PCR for the rapid detection of Candida and Aspergillus species in clinical specimens with improved sensitivity and specificity. Materials and Methods: A total of 71 proven cases of IFI (confirmed by culture) were collected. A total of 15 healthy, 15 patients suffering from bacterial sepsis and 15 patients with HIV, HBV viral infections were included as controls. Clinical specimens were subjected to a standardized nested amplification to produce Round I (504 bp) and Round II (150 bp) amplicons. Restriction digestion was performed on these products for further identification. Results: Analytical sensitivity was determined using 10⁶–10 CFU/ml of cell suspension. The lower detection limit of the assay was 10 CFU/ml of blood. This test was 100% sensitive and specific with a positive predictive value of 100% and a negative predictive value of 96.7%. Conclusion: The assay was found to be effective for the rapid detection of Candida and Aspergillus in clinical specimens.     

Application of PCR fingerprinting using (GACA)4 primer in the rapid discrimination of dermatophytes

Background: Superficial fungal infections have a major impact on cosmetic health, affecting more than 20-25% of the global population, which is predominantly caused by dermatophytes. As per literature search, molecular strain typing of dermatophytes has not been investigated in India. Therefore, the present study was carried out to characterise the dermatophyte species and strains by molecular methods. Objective: To analyse the genotype variability by applying polymerase chain reaction (PCR) fingerprinting using a simple sequence repetitive oligonucleotide (GACA)₄ primer to identify the species and strain variations among the dermatophytes isolated from a tertiary care centre in Chennai. Materials and Methods: From January 2010 to December 2010, 81 dermatophytes were isolated and included for the present study. A simple sequence repetitive oligonucleotide (GACA)₄ was used as a single primer in the amplification process. Results: The (GACA)₄-based PCR successfully amplified all the clinical isolates. Trichophyton rubrum and T. rubrum var. raubitschekii produced identical band profiles, where the latter could not be differentiated from the T. rubrum, which are being reported for the first time from south India. Epidermophyton floccosum produced species-specific band profiles. Intra-species variability was not observed among the T. rubrum and E. floccosum isolates. T. mentagrophytes produced three simple, distinct band patterns, which are surprisingly different from the earlier studies. Conclusion: The PCR-based genotype using the short primer is rapid and precise in direct identification of dermatophyte isolates by one-step PCR to the species level and strain discrimination of the T. mentagrophytes variants.     

Comparative evaluation of two polymerase chain reactions targeting different genomic regions to detect Mycobacterium tuberculosis in sputum

Purpose: Tuberculosis remains an important health problem all over the world, especially in resource poor settings like India. The Ziehl-Neelsen (ZN) staining of sputum smear is still the method of choice in the diagnosis of tuberculosis in spite of its low sensitivity and specificity. This paper evaluates comparison of two different polymerase chain reaction (PCR) assays with sputum smear findings to detect Mycobacterium tuberculosis. Materials and Methods: A total of 191 sputum samples were collected from 84 patients attending a tertiary care hospital, who were suspected of having pulmonary tuberculosis, were examined by PCR targeting two different genomic regions, namely, TRC₄ by non-nested format and IS6110 insertion element by nested format in comparison to ZN staining of sputum smears. Results: Among the patients tested, 20.24% (Mid-p 95%CI: 31.5–52.4) were smear positive, 7.14% (Mid-p 95%CI: 2.94–14.26) were positive by TRC₄ PCR and 41.67% (Mid-p 95%CI: 12.7–29.8) were positive by IS6110 nested PCR (nPCR). The median age of overall positive cases was 42 years. Among the nPCR positives, the median for age of rural and peri-urban community was 46 and 32 years, respectively. The kappa coefficient between smear findings and TRC4 PCR findings was 0.27 and an agreement of 0.83 was observed (Z = 2.99; one-tailed P = 0.001). TRC₄ PCR picked two unique positives that were negative by smear and IS6110 nPCR. Conclusion: The non-nested TRC₄ PCR showed inability for accurate detection of M. tuberculosis in sputum samples. The study concluded that the nPCR targeting IS6110 is superior and more sensitive than TRC₄ PCR.     

Free living amoebae in water sources of critical units in a tertiary care hospital in India

Background: Isolation of free-living amoebae (FLA) is reported sparsely from water taps, ventilators, air conditioners, haemodialysis units and dental irrigation systems of hospitals worldwide. Their prevalence in hospital environment especially in wards having immunocompromised patients may pose a risk to this group of susceptible population as they may cause disease themselves or may carry pathogens inside them. No study from India has performed such surveillance. Objective: To evaluate extent of FLA contamination in water sources of bone marrow transplant (BMT) intensive care unit (ICU), transplant ICU, haemodialysis unit and high dependency unit in a tertiary care hospital in India. Materials and Methods: A total of hundred samples including fifty each of tap water samples and swabs from mouth of taps used for drinking, bathing and hand washing purposes in these units were collected according to standard procedure. Samples were inoculated onto non-nutrient agar plates at room temperature followed by morphological confirmation. Molecular identification including polymerase chain reaction (PCR) and sequencing was performed in culture positive samples. Results: Four tap water samples and ten swab samples showed growth of trophozoites and cyst formation. Morphologically, four amoebae resembled Acanthamoeba spp. which was further confirmed by PCR and sequencing showed them to be of T3 and T4 genotypes. Conclusion: The presence of these FLA in hospital water sources emphasises the urgent need of implementing effective preventive measures. Further studies are required to estimate the true prevalence of FLA in Indian hospitals by taking larger number of samples.     

Haemophilus influenzae and SARS-CoV-2: Is there a role for investigation?

During the current pandemic of COVID-19, the authors observed that during screening test for SARS-CoV-2 targeting the E-gene by qRT-PCR, few nasopharyngeal/oropharyngeal samples showed amplification signals at late cycle threshold (C_T-value) > 35 despite being negative for other confirmatory target genes. Thirty such samples (taken as cases) showing detectable C_T of > 35 cycle in E-gene which were negative for other target genes of SARS-CoV-2 and 30 samples with undetectable fluorescence in E-gene were taken as controls for investigation. An in-vitro diagnostic approved commercial qRT-PCR multiplex kit detecting 33 respiratory pathogens which can also detect Haemophilus influenzae was used for screening the samples. It was observed that out of the 30 samples showing detectable C_T> 35 in E-gene, 11 samples were positive for Haemophilus influenzae whereas in the controls only three samples were positive for H. influenzae (p-value: 0.03) which was statistically significant. Further, the probes and primers were screened against H. influenzae for matches in the genome. It was observed that all primers and probes for the E-gene of SARS-CoV-2 had over 13 bp long sequences matching 100% with multiple sites across the H. influenzae genome. This qRT-PCR primer & probes are being used extensively across India, and laboratories using them should be aware of the cross-reactivity of primers & probes with the H. influenzae genome. Further, the authors observed that 95.9% (5415/5642) of COVID-19 positive cases detected in their laboratory were asymptomatic at the time of collection of samples. This warrants further investigations.