Experience of Indian association of medical microbiology external quality assurance scheme centre, New Delhi: Challenges and quality assessment of clinical microbiology laboratories

Introduction: EQAS program at New Delhi under IAMM was started in January 2014 across North and North east regions of India with 217 participants, which grew up to 540 by 2018. Materials and Methods: In 2014, 4 analytes per year were sent for 3 exercises, i.e. smear culture and serology. 2018 onwards PT analytes were increased from 4 to 12 and comparative performance of techniques analysed. Results: Out of the 22 smears sent for gram staining, ZN staining, Kinyoun staining and Albert staining, completely correct results ranged between 29.55% - 79.9%, 94.3% - 99.2%, 35.5% & 93.8%, respectively. Correct results for culture isolate identification & susceptibility testing and serology exercises varied between 70 & 92.4% and 73.1 & 98.59%, respectively. In the year 2018, 470 responses were received for bacterial culture identification & antibiotic susceptibility testing out of which manual and automated systems were used by 54% & 46% and 52.5% & 47.5% participants, respectively. Techniques used in BBV assays for HBsAg, HCV & HIV found all methods like ELISA, ELFA, CLIA and Card Test performing similarly. The major challenges in running the EQA program included requirement of large amount of specimens for PT item preparation, stability in hot and humid conditions and timely delivery of PT challenges in remote parts of the country. Conclusion: A large number of the participating laboratories (77%) had an overall score of >80% for all exercises, demonstrating acceptable baseline performance of EQAS registered laboratories. However, continued EQAS participation could further improve the quality of results.

Nucleic acid amplification test: Bridging the gap in blood safety & re-evaluation of blood screening for cryptic transfusion-transmitted infection among Indian donors

Background & objectives: Nucleic acid amplification test (NAT) in blood donor screening not only detects window period (WP) donors but also those with chronic occult infections which are negative by routine serological screening. This study was conducted to determine the time trend of NAT positivity and seroprevalence of transfusion-transmitted infections (TTIs) through a period of six years and evaluate the strength of NAT as a supplementary test in identifying the cryptic carriers in blood donor population. Methods: A total of 1,01,411 blood donations were screened between January 2011 and December 2016 by the ELISA and individual donor (ID) NAT Procleix Ultrio Plus Assay. Additional molecular and serological assays were done on the NAT yield samples to differentiate the type of cryptic carriers. Results: NAT yields comprised 0.05 per cent (50/101411) of the total samples tested with a yield rate of 1/2028. Hepatitis B virus (HBV) contributed to 80 per cent of the total NAT yields and the rest 20 per cent due to hepatitis C virus (HCV). Majority of HBV NAT yields (75%) were from chronic occult donors and 25 per cent were WP donors. Both HBV and HCV NAT yields had a wide range of viral count. There was no HIV NAT yield. A significant decline in the prevalence rate of TTIs through the study period of six years was observed. Interpretation & conclusions: The cryptic infections found in blood donors increase the risk of TTIs. Blood screening by both serology and NAT can reduce this threat.

Dominance of international high-risk clones in carbapenemase-producing Pseudomonas aeruginosa: Multicentric molecular epidemiology report from India

Background: Pseudomonas aeruginosa is one of the most common opportunistic pathogens that cause severe infections in humans. The burden of carbapenem resistance is particularly high and is on the rise. Very little information is available on the molecular mechanisms and its clonal types of carbapenem-resistant P. aeruginosa seen in Indian hospitals. This study was undertaken to monitor the ?-lactamase profile and to investigate the genetic relatedness of the carbapenemase-producing (CP) P. aeruginosa collected across different hospitals from India. Materials and Methods: A total of 507 non-duplicate, carbapenem-resistant P. aeruginosa isolated from various clinical specimens collected during 2014–2017 across seven Indian hospitals were included. Conventional multiplex polymerase chain reaction for the genes encoding beta-lactamases such as extended-spectrum beta-lactamase (ESBL) and carbapenemase were screened. A subset of isolates (n = 133) of CP P. aeruginosa were genotyped by multilocus sequence typing (MLST) scheme. Results: Of the total 507 isolates, 15%, 40% and 20% were positive for genes encoding ESBLs, carbapenemases and ESBLs + carbapenemases, respectively, whilst 25% were negative for the ?-lactamases screened. Amongst the ESBL genes, blaVEB is the most predominant, followed by blaPER and blaTEM, whilst blaVIM and blaNDM were the most predominant carbapenemases seen. However, regional differences were noted in the ?-lactamases profile across the study sites. Genotyping by MLST revealed 54 different sequence types (STs). The most common are ST357, ST235, ST233 and ST244. Six clonal complexes were found (CC357, CC235, CC244, CC1047, CC664 and CC308). About 24% of total STs are of novel types and these were found to emerge from the high-risk clones. Conclusion: This is the first large study from India to report the baseline data on the molecular resistance mechanisms and its association with genetic relatedness of CP P. aeruginosa circulating in Indian hospitals. blaVIM- and blaNDM-producing P. aeruginosa is the most prevalent carbapenemase seen in India. Majority of the isolates belongs to the high-risk international clones ST235, ST357 and ST664 which is a concern.

Impact of informational feedback to clinicians on antibiotic‑prescribing rates in a tertiary care hospital in Delhi.

Context: Antimicrobial use has been associated with increasing antimicrobial resistance. There is an urgent need for judicious use of antimicrobials. Informational feedback has been shown to result in changes in behavioural practices of physicians in certain healthcare settings. We conducted this study to see if the passive informational feedback can reduce in antimicrobial usage in a tertiary care centre. Aims: The study was undertaken to evaluate if the feedback to clinicians on their own antibiotic prescription results in any change in their antibiotic prescription habits. Settings and Design: The study was conducted at a tertiary care setting involving 33 units of different specialties. These units were split into 10 groups based on specialty and were allocated randomly to the control (16 units) and intervention (17 units) arms of the study. This study was a prospective intervention to assess the effect of prescribing feedback on clinical prescribing practices. Materials and Methods: In the intervention arm, information on resistance rates and antibiotic‑prescribing patterns was provided to all doctors. Behavioural change was assessed by comparing baseline prescribing rates of each unit with prescribing rates after the intervention. In the control arm, only information on monthly resistance rates was provided. Statistical Analysis: Change in the antimicrobial prescribing rates in the treatment group was assessed by using a Student’s t‑test. Results: The mean antibiotic use for all the specialties was 189 DDDs/100BDs. The prospective intervention did not elicit any effect on the antibiotic prescribing practices of the physicians. Low prescribers continued to prescribe antibiotics at a low rate, and high prescribers continued to prescribe at a high rate. Conclusions: In view of unfavourable results of passive intervention in the above study, active intervention may be more effective.

Comparative evaluation of two rapid Salmonella‑IgM tests and blood culture in the diagnosis of enteric fever.

Purpose: Enteric fever is a major public health problem in developing countries like India. An early and accurate diagnosis is necessary for a prompt and effective treatment. We have evaluated the diagnostic accuracy of two Rapid Salmonella‑IgM tests (Typhidot‑IgM and Enteroscreen‑IgM) as compared to blood culture in rapid and early diagnosis of enteric fever. Materials and Methods: A total of 2,699 patients’ serum samples were tested by Rapid Salmonella‑IgM tests and blood culture. Patients were divided into two groups. Test group—patients with enteric fever and blood culture positives for Salmonella Typhi; and three types of Controls, i.e. patients with non‑enteric fever illnesses, normal healthy controls and patients positive for S. Paratyphi‑ A. In addition to this we have also evaluated the significance of positive Salmonella‑IgM tests among blood culture‑negative cases. Results: The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Typhidot‑IgM test and Enteroscreen‑IgM test considering blood culture as gold standard were 97.29% and 88.13%, 97.40% and 87.83%, 98.18% and 92.03%, 96.15% and 82.27%, respectively. Typhidot‑IgM test was found to be significantly more sensitive and specific as compared to Enteroscreen‑IgM. Among blood culture‑negative patients, Rapid Salmonella‑IgM tests detected 72.25% additional cases of enteric fever. Although the Rapid Salmonella‑IgM tests are meant to diagnose S. Typhi only, but these tests detect S. Paratyphi‑ A also. Thirty‑eight patients who were blood culture‑positive for S. Paratyphi‑ A were also positive by Rapid Salmonella‑IgM tests. Conclusion: Rapid Salmonella‑IgM tests offer an advantage of increased sensitivity, rapidity, early diagnosis and simplicity over blood culture.

Utility of routine real time quantitative PCR monitoring of HCV infection in haemodialysis patients.

Background: Hepatitis C virus (HCV) infection causes signifi cant morbidity and mortality in patients of end stage renal disease (ESRD) on haemodialysis (HD). Stringent screening methods can help in its early diagnosis. Objective: The study addresses the utility of real-time quantitative polymerase chain reaction (RQ-PCR) in the diagnosis and monitoring of HCV infection especially on seronegative and normal serum alanine aminotransferase (ALT) HD patients. Material and Methods: This retrospective study was carried out from January 2010 to December 2012. Patients of ESRD on maintenance HD and on whom all the three assays HCV antibody serology, PCR and ALT were done were included in the study (n = 123). Group 1 (n = 57), comprised of patients with negative serology and normal ALT, and Group 2 (n = 66), had either raised ALT and or a positive or equivocal serology. Results: Out of the 123 cases studied, HCV serology was positive in 36.5% (45), ALT raised in 18.6% (23) and PCR positive in 67.4% (83) cases. PCR positivity was signifi cantly higher than serology and raised ALT. Group 2 had a signifi cantly higher PCR positivity than Group 1 (P = 0.0004), but 50.9% patients of Group 1, were also PCR positive and 69% of them had a high viral count of >8 × 105 IU/ml at the time of detection. Conclusion: Regular routine screening of HCV by RQ-PCR in ESRD patients can help in early diagnosis of HCV infection in patients with low index of suspicion.

Variables affecting the performance of galactomannan assay in high-risk patients at a Tertiary Care Centre in India.

Background: Diagnosis of invasive aspergillosis (IA) in immunocompromised patients using galactomannan ELISA (GM-ELISA) has shown variable sensitivity and specificity. Objectives: To assess the diagnostic performance of GM-ELISA and analyze the effect of decreasing the cut off value, neutropenia, antifungals and piperacillin-tazobactam (PTZ). Prognostic value using 30 day all-cause mortality was also determined. Materials and Methods: Serum samples from 81 patients categorized into "proven," "probable," and "possible," categories based on revised EORTC/MSG definitions were tested by GM-ELISA. Results: Sensitivity of GM-ELISA in proven, probable and possible cases was 91.7%, 84.6% and 83.3% respectively. At an index cut-off value of 0.5 an increased sensitivity with minimal loss of specificity was observed. Use of antifungals demonstrated a decrease in sensitivity in proven and possible cases whereas it remained unaffected in probable category. Specificity increased from 75% to 100% with a positivity criterion of >2 consecutive samples. Although an increase in specificity was observed in patients not receiving PTZ, it was not statistically significant. Serial GM index values increased significantly in neutropenic patients and were associated with a poor prognosis. Conclusions: GM-ELISA may be a useful diagnostic and prognostic modality for the detection of IA in high risk patients.

Use of John Cunningham virus polymerase chain reaction in the diagnosis of progressive multifocal leucoencephalopathy - A rare presenting manifestation in an HIV-positive patient.

John Cunningham virus infection is an important cause of progressive multifocal leucoencephalopathy (PML) in the context of advanced human immunodeficiency virus infection. Limited data are available regarding the true incidence of PML as a presenting manifestation of HIV. We report one such case and also highlight the effective use of polymerase chain reaction in confirming its diagnosis.

Utility of GenoType MTBDRplus assay in rapid diagnosis of multidrug resistant tuberculosis at a tertiary care centre in India.

Purpose: Molecular methods which allow rapid detection of tuberculosis as well as drug resistance directly from clinical samples have become the most popular diagnostic methodology with the emergence of multidrug resistant tuberculosis. The aim of the present study was to evaluate the performance of a line probe assay, GenoType MTBDRplus for the rapid detection of Mycobacterium tuberculosis and mutations causing rifampicin and INH resistance directly in smear positive pulmonary specimens and also in M. tuberculosis isolates grown from various clinical specimens. Materials and Methods: The MTBDRplus assay was done directly on 37 smear positive pulmonary specimens and also on 69 M. tuberculosis isolates obtained by rapid automated culture using Bact/Alert 3D. The results were compared with phenotypic drug susceptibility testing (1% proportion method) using Bact/Alert 3D. Results: The sensitivity and specificity for detection of resistance to rifampicin was 100% and 97.3%, and to INH was 91.9% and 98.4%, respectively, in comparison with the phenotypic drug susceptibility testing. Conclusion : MTBDRplus assay had good sensitivity and specificity with turn around time of less than 48 hours. It may be a useful tool for rapid detection of multidrug resistant tuberculosis at a tertiary care centre.

Clinical Pulmonary Infection Score to Diagnose Ventilator-associated Pneumonia in Children.

Background: There is a need to validate and suggest easy clinical method for diagnosis of ventilator-associated pneumonia (VAP) in developing countries. Objectives: To validate the use of simplified Clinical Pulmonary Infection Score (CPIS) for the diagnosis of VAP. Design: Prospective study. Setting: Pediatric intensive care unit of a tertiary care teaching hospital. Subjects: 30 children receiving mechanical ventilation for more than 48 hours and with simplified CPIS6. Methods: All patients underwent flexible bronchoscopy to obtain bronchoalveolar lavage which was analyzed quantitatively. Colony count 104cfu/mL was considered reference standard for definite VAP. Results: Of the five variables used for simplified CPIS, only patient’s temperature (P=0.013) and PaO2/ FiO2 ratio were significant (P<0.001) to differentiate the presence of definite VAP. Patients with definite VAP (BAL colony count 104cfu/mL) had CPIS of 8.4 while in no definite VAP group it was 6.4 (P= 0.007). CPIS of 8 was found to have sensitivity of 80%, specificity 80%, PPV 86.9%, NPV 70.5% and accuracy 80%. The area under Receiver operating characteristic curve of CPIS against reference standard was 0.81± 0.069 (P=0.001). Conclusion: Simplified CPIS is useful in patients on mechanical ventilation to diagnose ventilator- associated pneumonia.

Dengue NS1 antigen detection: A useful tool in early diagnosis of dengue virus infection.

Purpose: This study was undertaken to evaluate the efficacy of NS1 antigen (Ag) assay as an early marker for dengue virus (DV) infection. Materials and Methods: Group I evaluated the performance of NS1 antigen (Ag) assay in comparison to MAC-ELISA and their detection rate when performed together in a single sample. Six hundred acute/early convalescent sera were screened by both the assays. Group II evaluated the efficacy of a single assay in 30 acute phase sera of paediatric OPD patients screened only by NS1 Ag assay. Group III evaluated the specificity of NS1 assay in comparison to MAC-ELISA on 40 samples included as controls. Results: In Group I, 140 (23.3%) and 235 (39.1%) samples were positive by NS1 assay and MAC-ELISA respectively. The detection rate increased to 320 (53.3%) when both the assays were used together on a single sample. NS1 Ag positivity varied from 71.42% to 28.4% in acute and early convalescent sera, conversely IgM detection rate was 93.61% and 6.38% in early convalescent and acute phase sera respectively (P < 0.0001). In Group II, 66.66% (20) samples were positive by NS1 assay. All the samples in Group III were negative showing 100% specificity of both the assays. Conclusion: NS1 Ag assay holds promise in early diagnosis of dengue infection. When used in combination with MAC-ELISA on a single sample it significantly improves the diagnostic algorithm without the requirement of paired sera.

High level ciprofloxacin resistance in Salmonella enterica isolated from blood.

PURPOSE: Over the last few years, resistance to ciprofloxacin in Salmonella enterica has become a global concern. The present study was undertaken to find out the susceptibility pattern of Salmonella enterica isolates in our hospital. METHODS: Blood cultures were done using BacT/ALERT 3D system. The antimicrobial susceptibility testing was carried out by the Kirby-Bauer disc diffusion method using CLSI breakpoints. Minimum inhibitory concentration was determined for ciprofloxacin-resistant strains using E-test and Vitek-1 automated system. RESULTS: A total of 25,953 samples of blood culture yielded 431 Salmonella enterica serotype Typhi and 198 serotype Paratyphi A isolates. Twenty-two isolates of serotype Typhi were resistant to ciprofloxacin, while two isolates of Typhi and two Paratyphi A were intermediately susceptible to ciprofloxacin. Ciprofloxacin resistance is 5.6% (24 isolates) among Salmonella enterica serotype Typhi. Ampicillin, chloramphenicol and co-trimoxazole resistance in Salmonella enterica serotype Typhi appears to have decreased to 14.9% (64/431) in comparison to the 27% (55/205) during 2003. All isolates were sensitive to ceftriaxone. CONCLUSIONS: Ciprofloxacin can no longer be considered as the drug of choice in treating Salmonella infections. While first-line antimicrobials may still have a role to play in the treatment of enteric fever, ceftriaxone remains the sole defence against ciprofloxacin-resistant Salmonella infections.

Nasopharyngeal carriage of Streptococcus pneumoniae.

OBJECTIVE: To determine nasopharyngeal carriage rate and prevalent serogroups/types (SGT) of S. pneumoniae in healthy children, assess their antimicrobial susceptibility and its implications over the heptavalent pneumococcal conjugate vaccine. METHODS: 200 healthy children aged between 3 months and 3 years attending Pediatric OPD at Sir Ganga Ram Hospital, New Delhi were studied. A nasopharyngeal swab was collected from each child which was processed to isolate Streptococcus pneumoniae. Serotyping was performed by the Quellung reaction. Antimicrobial susceptibility patterns were determined by disk diffusion and E test methods. RESULTS: S. pneumoniae carriage rate was 6.5%. Isolates belonged to serotypes 1, 6, 14 and 19, of which serotype 19 was the most common. None of the strains were totally resistant to penicillin though 2 (15.4%) were intermediately resistant. Overall, 84.6% of the isolates belonged to the strains covered by the heptavalent pneumococcal vaccine. CONCLUSION: The heptavalent conjugate vaccine covers most isolated strains, but since the number of strains is very small, it is suggested that there is need for further studies in different regions to assess the usefulness of this vaccine.

Vertical transmission of Salmonella paratyphi A.

Neonatal enteric fever is a rare but life-threatening illness. Patients may present with varying severity, Salmonella enterica serotype Typhi causing more severe illness than Salmonella enterica serotype Paratyphi A. Salmonella enterica serotype Paratyphi A is considered to cause milder infection with fewer complications. We report a rare case of vertical transmission of Salmonella enterica serotype Paratyphi A with severe complications and high mortality. Even though there are case reports of vertical transmission of Salmonella enterica serotype Typhi, to our knowledge, this is the first case report of vertical transmission of Salmonella enterica serotype ParatyphiA. The role of blood culture in accurate diagnosis and treatment is also discussed.

Cutaneous cryptococcosis.

We report a case of an apparently immunocompetent male, who presented with a painless nodule over the upper abdominal wall. He gave a history of exposure to pigeon droppings. Cryptococcus neoformans was isolated from the lesion and no underlying disorder could be detected. He improved on treatment with oral Fluoconazole.

Mixed salmonella infection - A case report.

Mixed infection with multiple Salmonella serotypes in the same patient is an unusual finding. We present a case of enteric fever in which the blood culture was sterile and Widal test was negative. The culture of the bone marrow yielded Salmonella typhi and Salmonella paratyphi A.