Laboratory detection of bacterial pathogens and clinical and laboratory response of syndromic management in patients with cervical discharge: A retrospective study

Background: Cervical discharge as part of cervicitis and pelvic inflammatory disease is a cause of significant morbidity in sexually active women worldwide. Non-gonococcal and non- chlamydial bacterial pathogens are becoming more prevalent. Aims: This study aims to determine bacterial pathogens causing cervical discharge using culture and/or polymerase chain reaction and assess the clinical and laboratory response to the conventional syndromic kit regimen established by the World Health Organisation. Methods: A retrospective review of records of women with cervical discharge over one year period. Culture and/or polymerase chain reaction results of endocervical swabs of various bacterial pathogens at baseline and after four weeks of treatment with syndromic kit regimen were recorded. Results: A total of 70 case records were reviewed for clinical details, out of which results of bacterial culture and polymerase chain reaction were available for 67 cases. Infectious aetiology was found in 30 (44.7%) patients with Ureaplasma species being the most common organism isolated on culture (18, 26.8%) and polymerase chain reaction (25, 37.3%), respectively. Polymerase chain reaction for Chlamydia trachomatis and Mycoplasma hominis was positive in ten (14.9%) and four (6%) cases, respectively. None of the patients showed positive culture for Neisseria gonorrhoeae. Coinfection was seen in eight (11.9%) patients with the majority showing Chlamydia trachomatis and Ureaplasma spp. coinfection (five patients). Forty one cases (58.5%) received tab. cefixime 400 mg and tab. azithromycin one gram stat (kit 1), while 29 cases (43.3%) received tab. cefixime 400 mg stat, tab. metronidazole 400 mg and cap. doxycycline 100 mg, both twice daily for 14 days (kit 6). Minimal to no clinical improvement with treatment was seen in 14 out of 32 cases (44%) at the end of four weeks with the conventional kit regimen. Post-treatment culture and/or polymerase chain reaction were positive in nine out of 28 cases (32.1%) with Ureaplasma spp. being the most common. Limitations: Retrospective study design, small sample size and fewer cases with follow-up data were the main limitations. Conclusion: Ureaplasma spp. was the most common infectious cause of cervical discharge in our patients. Treatment given as part of syndromic management led to a clinical and microbiological response in around half and two-third cases, respectively.

A pilot study to determine Neisseria gonorrhoeae–Chlamydia trachomatis coinfection rates in symptomatic patients attending STI Clinics, New Delhi, India

Background: Neisseria gonorrhoeae and Chlamydia trachomatis are the two most prevalent bacterial sexually transmitted infections. For over two decades, treatment guidelines have recommended empirical co-treatment for N.gonorrhoeae and C.trachomatis as symptoms overlap and co-infection is common. Studies from India estimating the same are limited and mostly based on conventional techniques. Aim and Objective: The aim of this study was to determine the frequency of N.gonorrhoeae and C.trachomatis coinfection using nucleic acid amplification tests. Further, we assessed the utility of pus cell estimation in Gram stained smears as a screening tool for inclusion of samples for molecular diagnosis. Methods: This was a prospective study conducted at two tertiary care hospitals; 100 patients (55 females and 45 males) with genitourinary discharge attending STI clinics were recruited, and endocervical or urethral swabs were collected. PCRs for N.gonorrhoeae and C.trachomatis were put up. In addition, microscopy and culture for gonococcus was performed followed by antimicrobial susceptibility testing. Statistical analysis was performed using the SPSS 16 software. Results: N.gonorrhoeae infection was more common than C.trachomatis. A total of 14 patients were positive by PCR (9 males and 5 females) for gonococcus. However, culture was positive only in 8 male patients. PCR for C.trachomatis was positive in 9 (4 males and 5 females) and the co-infection rate was 5%. The sensitivity and negative predictive value of pus cell estimation was 100% for males and 64% and 94.6% respectively for females. All isolates were susceptible to extended spectrum cephalosporins and azithromycin. Limitation: The sample size of the study was small. Conclusion: Frequency of N.gonorrhoeae/C.trachomatis coinfection in symptomatic STI patients is low. Coinfection is considerably overestimated and necessary confirmation of etiological diagnosis could reduce widespread empirical administration of broad-spectrum antibiotics.

Risk Factors for Central line – associated Bloodstream Infections.

Objective: To carry out surveillance of central line – associated bloodstream infections in a Pediatric intensive care unit (PICU) and determine associated risk factors. Methods: This prospective study was conducted over 1.5 years in the PICU. CDC definitions for these infections were followed and associated risk factors were identified. Results: Of 265 enrolled children with central line, 13 developed blood stream infections (incidence density 5.03/1000 central-line days). Significant risk factors included changing the central-line, especially triple lumen, and frequently accessing the central-line. Conclusion: Central-line associated bloodstream infections are preventable primary bacteremias and intervention strategies for prevention should be based on evidence generated to devise future protocols.

Nucleic acid amplification tests (NAATs) for gonorrhoea diagnosis in women: Experience of a tertiary care hospital in north India.

Background & objectives: Gonorrhoea is among the most frequent of the estimated bacterial sexually transmitted infections (STIs) and has significant health implications in women. The use of nucleic acid amplification tests (NAATs) has been shown to provide enhanced diagnosis of gonorrhoea in female patients. However, it is recommended that an on-going assessment of the test assays should be performed to check for any probable sequence variation occurring in the targeted region. In this study, an in-house PCR targeting opa-gene of Neisseria gonorrhoeae was used in conjunction with 16S ribosomal PCR to determine the presence of gonorrhoea in female patients attending the tertiary care hospitals. Methods: Endocervical samples collected from 250 female patients with complaints of vaginal or cervical discharge or pain in lower abdomen were tested using opa and 16S ribosomal assay. The samples were also processed by conventional methods. Results: Of the 250 female patients included in the study, only one was positive by conventional methods (microscopy and culture) whereas 17 patients were found to be positive based on PCR results. Interpretation & conclusions: The clinical sensitivity of conventional methods for the detection of N. gonorrhoeae in female patients was low. The gonococcal detection rates increased when molecular method was used giving 16 additional positives. Studies should be done to find out other gene targets that may be used in the screening assays to detect the presence of gonorrhoea.

Induction of resistant mutants of Salmonella enterica serotype Typhi under ciprofloxacin selective pressure.

Background & objectives: Infection with Salmonella enterica serovar Typhi (hereafter S. Typhi) is an important public health problem in India. There has been an increase in the number of reported clinical failures to ciprofloxacin treatment but the data on possible mechanism of failure are limited. One mechanism that has been widely reported and found associated with ciprofloxacin resistance, is the mutations in target genes in QRDR (quinolone resistance determining region). It is hypothesized that mutations in DNA gyrase or topoisomerase IV result in therapeutic failure under selective pressure of antibiotic while the patient is on treatment. We undertook in vitro sequential selection studies to expose the clinical isolates of S. Typhi to different concentration of ciprofloxacin to study the role of antibiotic selective pressure in the development of mutations in QRDR. Methods: Total 26 clinical isolates were divided in to two parts: part I included six isolates obtained from three patients with relapse of enteric fever and part II included 20 isolates with different ciprofloxacin MIC levels. For in vitro induction of mutation experiment, five S. Typhi isolates were selected which included three NAS (nalidixic acid sensitive) and 2 NAR (nalidixic acid resistant) S. Typhi. These isolates were grown under increasing concentrations of ciprofloxacin and mutations acquired in QRDR of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) were investigated by sequencing. Results: For the isolates included in the part I of the study, it was found that the MIC to ciprofloxacin increased in the isolates obtained during the relapse of enteric fever as compare to the first isolate. All isolates had single mutation in gyrA gene at S83 without additional mutation in the second isolate. In the second part of the study, the nine isolates with varying MICs to ciprofloxacin also had single mutation in gyrA gene at S83 and another six had triple mutations, two mutations in gyrA gene (at S83 and D87) and one mutation in parC gene (at S80). In in vitro induction of mutation experiment, all mutated isolates showed triple mutation (two mutation in gyrA and one in parC gene) while no mutations were found in wild isolates. Interpretation & conclusions: Upon exposure to the step-wise increased concentration of ciprofloxacin, isolates become more tolerant to the ciprofloxacin and showed 2-4 fold higher MICs without new mutation after 8 μg/ml. So the accumulation of mutations under continuous ciprofloxacin pressure and tolerance of the mutant isolates led to the clinical failure. These results also suggested that there could be another mechanism responsible for resistance.

Central venous catheter related bloodstream infections in medical intensive care unit patients in a tertiary referral centre.

Aims. To determine the incidence of central line associated bloodstream infections (CLABSIs) in the medical intensive care unit (ICU) and ward setting at All India Institute of Medical Sciences (AIIMS), New Delhi. Settings and Design. The study was conducted in the medical ICU, a 9-bedded ICU at the AIIMS, a tertiary care teaching hospital. The study design was a prospective observational study. Methods. One hundred patients admitted to medical ICU and the ward at AIIMS with an indwelling, non-tunnelled central venous catheter (CVC) in place at admission and those with a hospital stay with indwelling CVC for more than 48 hours were monitored. These patients were followed daily for the development of new onset sepsis 48 hours after insertion of CVC, in which case three sets of blood samples for culture were drawn over a span of 24 hours. Statistical Methods. Incidence of CLABSIs was measured per 1000 central line days. Results. One hundred patients hospitalised for an aggregate 1119 days acquired 29 hospital-acquired infections (HAIs), a rate of 38.8% or 31.2 HAIs per 1000 hospital days. The incidence of bloodstream infections (BSIs) in this group was 6.8%. No case of laboratory confirmed CLABSIs could be demonstrated. Incidence of clinical sepsis was 27.6% or 8.2 per 1000 CVC days. There were 9 cases out of the 29 patients (39.7%) who had evidence of HAIs with no apparent focus of infection. Only one of these cases had evidence of BSI with isolation of Staphylococcus aureus in both CVC tip culture and the simultaneous blood culture; however the antibiograms were different. Conclusions. The low rate of BSIs in the present study and the absence of occurrence of a laboratory confirmed CLABSI should be interpreted in the light of the small sample size of the study and the multitude of antibiotics received before the development of HAI.

Prospective study estimating healthcare associated infections in a paediatric hemato-oncology unit of a tertiary care hospital in north India.

Background & objectives: Healthcare associated infections (HAIs) are responsible for morbidity and mortality among immunocompromised and critically ill patients. We undertook this study to estimate the burden of HAIs in the paediatric cancer patients in a tertiary care hospital in north India. Methods: This prospective, observational study, based on active surveillance for a period of 11 months was undertaken in a 4-bedded isolated, cubicle for paediatric cancer patients. Patients who stayed in the cubicle for ≥48 h, were followed prospectively for the development of HAIs. Results: Of the 138 patients, 13 developed 14 episodes of HAIs during the study period. Patient-days calculated were 1273 days. Crude infection rate (CIR) and incidence density (ID) of all HAIs were 9.4/100 patients and 11/1000 patient-days, respectively. Of the 14 episodes of HAIs, seven (50%) were of blood stream infections (HA-BSI), five (36%) of pneumonia (HAP) and two (14%) urinary tract infections (HA-UTI). The CIRs of HA-BSI, HAP and HA-UTI were 5.1, 3.6 and 1.4/100 patients, respectively. The corresponding IDs were 5.5, 3.9 and 1.6/1000 patient-days, respectively. Mean length of stay was significantly higher in patients who developed an HAI [13.8 (range 7-30), median (Interquartile range) 12 (11-14)] vs 7.5 days [range 2-28, median (interquartile range) 7 (5-9); P<0.0001]. Also mortality was significantly higher in patients who developed an HAI [23% (3/13) vs 3% (4/125), P<0.05]. Interpretation & conclusions: The incidence of HAIs in the paediatric cancer patients in the study was 11/1000 patient days, of which HA-BSIs were the commonest. HAIs were associated with an increase in morbidity and mortality amongst this high risk patient population.

Genital Chlamydia trachomatis: An update.

Chlamydia trachomatis is the most common cause of curable bacterial sexually transmitted infection (STI) worldwide. It manifests primarily as urethritis in males and endocervicitis in females. Untreated chlamydial infection in man can cause epididymitis and proctitis. Though most women with Chlamydia infection are asymptomatic or have minimal symptoms, some develop salpingitis, endometritis, pelvic inflammatory disease (PID), ectopic pregnancy and tubal factor infertility. It is associated with an increased risk for the transmission or acquisition of HIV and is also attributed to be a risk factor for the development of cervical carcinoma. Early diagnosis and treatment of infected individuals is required to prevent the spread of the disease and severe sequelae. Traditionally, tissue culture was considered the gold standard for the diagnosis. However, with the availability of newer diagnostic techniques particularly molecular methods which are not only highly sensitive and specific but are cost-effective also, the diagnosis has became fast and easy. The purpose of this review is to study the various aspects of genital C. trachomatis infection. Also the advances related to the clinical picture, various diagnostic modalities, prevention, treatment, drug resistance and control measures will be dealt with.

Multiple locus sequence typing of Salmonella Typhi, isolated in north India - a preliminary study.

Background & objectives: In India enteric fever is a major public health problem and Salmonella Typhi is the most common aetiologic agent. Any control strategy for such infections depends to a large extent on the understanding of the disease and relatedness of strains across the world. Multi locus sequence typing (MLST) is one such method of genotyping of bacteria based upon housekeeping genes of known function and chromosome position. MLST data of pathogens are important to determine the molecular evolution by a stable and reproducible method. This study was undertaken to determine the sequence types of representatives S. Typhi isolates obtained from enteric fever patients in a tertiary care centre in north India, over a period of 20 years (1990-2010). Methods: A total of 30 representative isolates of S. Typhi identified by biochemical and serological tests were subjected to multi locus sequence typing (MLST). Seven housekeeping genes of known function and chromosome position were used for the typing by MLST. Sequencing was carried out by using an automated DNA sequencer and results were analyzed to generate phylogenetic tree. Results: MLST pattern grouped S. Typhi into two sequence types- ST1 and ST2. ST1 was predominantly present followed by ST2. Interpretation & conclusions: By MLST the presence of both sequence types, ST1 and ST2, was found in S. Typhi isolates in our region. Predominately ST1 was present followed by ST2. These preliminary results corroborate the global distribution of both sequence types of S. Typhi and also emphasize for the continuous screening of S. Typhi.

Characterization of nasopharyngeal isolates of type b Haemophilus influenzae from Delhi.

Background & objectives: Haemophilus influenzae is an important cause of mortality and morbidity among young children in developing countries. Increasing incidence of antibiotic resistance especially production of extended spectrum beta lactamase (ESBL) has made treatment and management of H. influenzae infection more difficult. Nasopharyngeal H. influenzae isolates are excellent surrogate for determination of antibiotic resistance prevalent among invasive H. influenzae isolates. In this study, we characterized nasopharyngeal H. influenzae isolates obtained from healthy school going children in Delhi. Methods: Nasopharyngeal H. influenzae isolates were collected from healthy school going children and subjected to serotyping, fimbrial typing and antibiogram profiling. ESBL production was recorded using phenotypic as well as molecular methods. Multi locus sequence typing (MLST) of 13 representative nasopharyngeal H. influenzae isolates was performed as per guidelines. Results: A significant proportion (26 of 80, 32.5%) of nasopharyngeal isolates of H. influenzae were identified as serotype b. Fimbrial gene (hifA) was detected in 23 (28.75%) isolates. Resistance against commonly prescribed antibiotics (Amp, Tet, Chloro, Septran, Cephalexin) were observed to be high among the nasopharyngeal commensal H. influenzae. Extended spectrum beta lactamase (ESBL) production was observed in a five (6.25%) isolates by both double disk diffusion and molecular typing. MLST identified several novel alleles as well as novel sequence types. Interpretation & conclusions: Our findings showed high resistance against common antibiotics and detection of ESBL in nasopharyngeal H. influenzae isolates collected from normal healthy school going children in Delhi. Detection of H. influenzae type b capsular gene and the presence of fimbrial gene (hif A) suggest virulence potential of these isolates. Discovery of novel alleles and presence of new sequence types (STs) among nasopharyngeal H. influenzae isolates may suggest wider genetic diversity.

A pilot study for diagnosis of genital Chlamydia trachomatis infections by polymerase chain reaction among symptomatic Indian women.

Background: Chlamydia trachomatis is the most common bacterial etiology of sexually transmitted infection. Aim : A pilot study was designed using PCR for amplification and detection of a specific 517 bp sequence of the common endogenous plasmid of C. trachomatis from clinical swab specimens obtained from symptomatic female patients attending STD clinics of AIIMS and Regional STD Teaching, Training & Research Center, Safdarjang Hospital, New Delhi. Methods: 97 patients were recruited in the study, and endocervical swabs were collected following standard procedures. The samples were analyzed by PCR and direct fluorescence antibody (DFA) for detection of C. trachomatis, and the sensitivity, specificity, PPV and NPV of PCR were calculated taking DFA as gold standard. Results: Out of 97 samples tested, 9 were positive for C. trachomatis by PCR. 1 PCR positive patient was negative by DFA although a total of 11 patients were positive by DFA. The sensitivity, specificity, PPV and NPV of PCR with reference to DFA was 72.73%, 98.84%, 88.89% and 96.59%, respectively. This PCR had high specificity and NPV for detection of C.trachomatis. Conclusions : In light of the introduction of enhanced syndromic approach, which involves the use of laboratory techniques (wherever possible) to confirm clinical diagnosis, a diagnostic PCR with high specificity and NPV is particularly valuable for determination of etiological diagnosis and hence contribute to judicious use of antimicrobials in the community.

Primary meningococcal polyarthritis in a young man.

We report a rare occurrence of primary meningococcal polyarthritis in a 19-year-old man. The fluid in the elbow joint showed Gram-negative diplococci but the culture was sterile. The diagnosis was confirmed by polymerase chain reaction targeting crgA gene of Neisseria meningitidis.

Interobserver variation in the interpretation of Nugent scoring method for diagnosis of bacterial vaginosis.

Background & objectives: Bacterial vaginosis (BV) is the most common form of vaginal infection and an important cause of morbidity in women of reproductive age. This study was carried out to examine the interobserver variation on interpretation of Nugent scoring method in the diagnosis of BV. Methods: This prospective study was conducted in a rural primary health care center of north India from May 2003 to April 2004 and included 601 married, sexually active women between 18-49 yr of age presented with self-reported symptoms of vaginal discharge and/or genital itching and/or genital burning. Specimens collected from the lateral wall of vagina were subjected to Gram staining and the microscope slide smears were examined by 3 independent observers. Each of the three observers scored and interpreted the slides for diagnosis of bacterial vaginosis using the Nugent method. Results: Complete agreement amongst the three observers was found in 76.2 per cent of cases. In 22.13 per cent cases, two observers were in agreement while interpretation of the slides were in complete disagreement only in 1.66 per cent of cases. The interrater reproducibility was found to be excellent between observers 1 and 3, while between observers 1 and 2, and 2 and 3 it was good to fair. Interpretation & conclusions: Nugent scoring system appears to be a reliable and convenient method for laboratory evaluation of cases of bacterial vaginosis. At the same time, one must be aware of the factors that might lead to discrepant results.

Enterococcal infections & antimicrobial resistance.

Enterococci have traditionally regarded as low grade pathogens, have emerged as an increasingly important cause of nosocomial infections in the last decade. Although about a dozen enterococcus species have been identified, only two are responsible for the majority of human infections, i.e., Enterococcus faecalis and E. faecium. The most common nosocomial infections produced by these organisms are urinary tract infections (associated with instrumentation and antimicrobial resistance), followed by intra-abdominal and pelvic infections. They also cause surgical wound infections, bacteraemia, endocarditis, neonatal sepsis and rarely meningitis. A major reason why these organisms survive in hospital environment is the intrinsic resistance to several commonly used antibiotics and, perhaps more importantly, their ability to acquire resistance to all currently available antibiotics, either by mutation or by receipt of foreign genetic material through the transfer of plasmids and transposons. The emergence of vancomycin-resistant enterococci (VRE) is a cause of concern, as once established, it is very difficult to control. Moreover, there can be transfer of resistant gene from enterococci to Staphylococcus aureus thereby posing a threat to the patient safety and also challenges for the treating physicians. This review highlights the shifting spectrum of enterococcal infections, along with their geographical distribution and growing nosocomial importance. Emergence of antimicrobial resistance, pathogenicity and virulence factors, current preventive, control and treatment modalities of severe enterococcal infections are also dealt with.

InPouch TV culture for detection of Trichomonas vaginalis.

BACKGROUND & OBJECTIVE: Trichomonas vaginalis accounts for almost half of all curable sexually transmitted infections and has also been associated with adverse outcomes of pregnancy and increased risk of HIV in women. Diagnosis of the condition by direct wet mount examination has a low sensitivity. Herein, we describe our experience with InPouch culture system for the detection of T. vaginalis. METHODS: This prospective study was carried out from May 2003 to April 2004 among women presenting with genitourinary symptoms attending a primary health center clinic in Ballabhgarh, India. Two vaginal swabs (cotton tips) were obtained from each woman. The first swab was obtained from the lateral wall of vagina and was used to make a wet mount preparation. The second swab was obtained from the posterior fornix of the vagina and inoculated in the InPouch for culture of T. vaginalis. RESULTS: Of the 601 women, 22 were positive by direct microscopy for T. vaginalis while 40 were positive by culture. Overall, T. vaginalis accounted for 6.7 per cent of reproductive tract infections. INTERPRETATION & CONCLUSION: The InPouch TV culture system is a simple, cost-effective and a sensitive method for diagnosing T. vaginalis and may be recommended for routine use in diagnosing genital tract infections.

Occurrence of TEM & SHV gene in extended spectrum beta-lactamases (ESBLs) producing Klebsiella sp. isolated from a tertiary care hospital.

BACKGROUND & OBJECTIVE: Extended-spectrum beta-lactamases (ESBLs) are rapidly evolving group of beta-lactamase enzymes produced by the Gram negative bacteria. These enzymes have been derived from TEM and SHV genes by mutations and have been well described in Klebsiella pneumoniae. Information on molecular types of ESBL positive Klebsiella sp. is lacking from India. We therefore undertook this study to look for the TEM and SHV genes in ESBL positive Klebsiella sp. isolated from the patients admitted to a tertiary care hospital in north India. METHODS: A total of 204 multidrug-resistant isolates of Klebsiellae obtained from clinical samples; blood (n=108), urine (n=15), pus (n=2) and sputum (n=79) were obtained and screened for resistance to 3rd generation cephalosporins (3GC). The ESBL status was determined by double disk diffusion test (DDDT) and further by ESBL E-test. Multiplex PCR specific for TEM and SHV genes was performed to distinguish four different genotypes: TEM-positive, SHV-positive, TEM- and SHV-positive and non-TEM non-SHV ESBL types. RESULTS: Eighty six per cent (175 of 204) of the isolates were found to be resistant to at least one of the 3GCs, of which 97.1 per cent (170) of Klebsiella sp. isolates were confirmed to be positive for ESBL. Of these 170 isolates, 95 were randomly selected for PCR of TEM and SHV genes. Isolates having both TEM and SHV genes were common (67.3%) whereas only 20 per cent isolates possessed TEM gene and 8.4 per cent SHV gene alone. INTERPRETATION & CONCLUSION: Our findings showed that the majority of the ESBL positive clinical isolates of Klebsiella sp. carried both TEM and SHV genes followed by TEM alone. Such studies need to be done in various geographical regions of the country to know about the prevalent genotypes for better management of infection.

Profile of bacterial isolates from suspected cases of infective endocarditis (IE): a retrospective study in a north Indian tertiary care hospital.

Infective Endocarditis (IE) is an emerging infection of the twenty-first century. This chronic Infection is mainly caused by bacteria, although fungi can also be associated with it. It is Important to know the profile of bacteria causing IE in a given region so as to suggest the empirical therapy for this serious illness. Blood culture isolates of clinically diagnosed or suspected cases of IE admitted to various wards of the All India Institute of Medical Sciences were analyzed retrospectively from January 2000 to June 2004. Standard techniques were used for the isolation and identification of the bacteria. Our study has demonstrated the predominance of Gram-negative bacilli, especially, Acinetobacter species and Pseudomonas aeruginosa, which are notorious for antimicrobial resistance, as the aetiological agents of IE. Amongst Gram-positive cocci, Enterococci exhibiting HLAR comprised the predominant species. Methicillin resistance among staphylococcal strains in this Tertiary care hospital is adding to the therapeutic challenge in the management of this serious illness. Although antimicrobial treatment should not be delayed in such cases, we cannot undermine the importance of isolation and identification of the etiological agents and the determination of the antimicrobial susceptibility for the management of these life-threatening conditions as well as for the formulation of guidelines for empirical therapy of these cases.