Meningococcal Meningitis: A Multicentric Hospital-based Study in Kathmandu, Nepal
Supriya Sharma1, *, Jyoti Acharya2, Dominique A. Caugant3, Jeewan Thapa4, Manju Bajracharya5, Madhusudan Kayastha6, Saroj Sharma6, Bimal S. Chalise7, Ragesh Karn8, Megha R. Banjara1, Prakash Ghimire1, Anjana Singh1
Identifiers and Pagination:Year: 2019
First Page: 273
Last Page: 278
Publisher Id: TOMICROJ-13-273
Article History:Received Date: 16/08/2019
Revision Received Date: 02/11/2019
Acceptance Date: 07/11/2019
Electronic publication date: 11/12/2019
Collection year: 2019
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The global epidemiology of meningococcal disease varies markedly by region and over time. In Nepal, information on serogroup of meningococci is not available since the 1983 serogroup A epidemic in Kathmandu.
To provide some fundamental data on the circulating serogroups of meningococci for potential meningococcal immunization programs in Nepal.
This cross-sectional prospective study was conducted from January 2017 to December 2018 among 387 clinically suspected meningitis cases. Cerebrospinal fluid samples were collected by lumbar puncture technique at five referral hospitals of Kathmandu and processed by conventional cultural techniques. Neisseria meningitidis was identified by colony morphology, Gram staining and oxidase test. Serogrouping of meningococci was performed by slide agglutination test. Antibiotic susceptibility testing was done by the modified Kirby Bauer disc diffusion method. The data was entered into IBM SPSS Statistics 21 software and a p-value of <0.05 was considered significant.
Thirty-two samples were positive by culture for a bacterial pathogen with 2.3% of meningococci. All except one meningococcal meningitis cases were aged below 15 years. All N.meningitidis isolates belonged to serogroup A and were susceptible to ceftriaxone, chloramphenicol, meropenem and minocycline; however, 22% isolates showed resistance to cotrimoxazole and 11% intermediate resistance to ciprofloxacin.
The circulating serogroup of N. meningitidis in Kathmandu has not changed over the past 35 years. The prevalence of meningococcal meningitis in Kathmandu is low but might be underestimated due to the sole use of culture-based diagnostic methods. Detection of meningococci by alternative methods may be useful in the precise estimation of actual disease burden.
Neisseria meningitidis is one of the leading causes of bacterial meningitis worldwide . The global epidemiology of meningococcal meningitis varies markedly by region and over time . The highest incidence occurs in the “meningitis belt” of sub-Saharan Africa . Epidemiological data on the burden of meningococcal meningitis in South-East Asia are scarce . In Nepal, a large serogroup A meningococcal outbreak occurred in the Kathmandu valley in 1982 . Since then, only a few studies have been published on meningococcal meningitis in Nepal [6, 7].
Of the twelve serogroups of N. meningitidis, 6 (A, B, C, W, X and Y) can cause epidemics . The global distribution of serogroups of meningococci is variable. Serogroups B and C are most common in America, Europe and Australia, whereas serogroup A has caused the majority of the disease in Asia, as well as in Africa, before the introduction of a monovalent conjugate vaccine in large mass campaigns starting in 2010. Sometimes, some serogroups can emerge in certain regions or countries like serogroup C in China and serogroup Y in North America . Two cases of serogroup B have been reported in India, a neighboring country of Nepal [8, 9].
Information on circulating serogroups of meningococci at the present time and in the local context is essential for the authorities to make evidence-based decisions whether to use vaccines or other modalities of control measures in the national program. In Nepal, meningococcal vaccination has not yet been introduced into the national immunization program. Therefore, this study was conducted to provide some fundamental data on the circulating serogroups of meningococci for potential meningococcal immunization program in Nepal.
2. MATERIALS AND METHODS
2.1. Study Design
This cross-sectional prospective study was conducted from January 2017 to December 2018 among clinically suspected meningitis cases attending five major hospitals located at Kathmandu, Nepal - Bhaktapur Hospital, Bir hospital, Kanti Children's Hospital (KCH), Sukraraj Tropical and Infectious Diseases Hospital (STIDH) and Tribhuvan University Teaching Hospital (TUTH).
2.2. Sample Size
Considering the estimated prevalence of bacterial meningitis (p) to be 7.2% with 95% confidence interval (z), 3% maximum tolerable error and 10% drop out, the minimum sample size calculated was 314 . Altogether, 387 samples were included in this study.
2.3. Sample Collection and Processing
Cerebrospinal Fluid (CSF) sample was collected by the attending physician/medical officer by lumbar puncture from each clinically suspected meningitis case at the respective study site. A sterile wide-bore needle was inserted between the lumbar vertebrae L4 and L5, and the CSF sample was allowed to drip into the sterile container. A loopful of specimen was inoculated immediately within 30 minutes into blood agar and chocolate agar (Hi-Media Laboratories, Pvt. Limited, India) plates and incubated in candle jar (5-10% CO2) at 37ºC for 24 hours.
2.4. Identification of Bacteria
Identification of bacterial isolates was done at National Public Health Laboratory, Teku, Kathmandu, by standard microbiological techniques including observation of colony characteristics, Gram staining, catalase, oxidase and other required biochemical tests. Additionally, bile solubility test and optochin sensitivity test were performed for identification of S. pneumoniae and X and V factor requirement test for H. influenzae.
Serogrouping of N. meningitidis isolate was done by slide agglutination test using group-specific antisera (Remel Europe Ltd, UK). Briefly, bacterial suspension was prepared by emulsifying 2-3 colonies of N. meningitidis in a drop of sterile normal saline on a disposable card. A drop of polyvalent antisera A-D and X-Z, W135 was added, appropriately mixed and rotated at 120 revolutions per minute (rpm). The appearance of any agglutination within 2 minutes was observed. If positive, it was tested for serogroups A, B, C, W and Y using group-specific monovalent antisera. N. meningitidis Z1503, Z5163, Z6432 and Z6434 were used as control strains .
Antibiotic susceptibility testing was done by the modified Kirby Bauer disc diffusion method following Clinical and Laboratory Standards Institute (CLSI) guidelines . Antibiotic discs (Hi-Media Laboratories, Pvt. Limited, India) used were ceftriaxone (30 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), meropemem (10 μg), minocycline (30 μg) and trimethoprim-sulfamethoxazole (1.25/23.75 μg). S. pneumoniae ATCC 49619, H. influenzae ATCC 49247, N.meningitidis Z1503 and Escherichia coli ATCC 25922 were used as control organisms for antibiotic susceptibility testing.
2.5. Data Analysis
The data were entered into Microsoft Office Excel 2007 and IBM SPSS Statistics 21 software. Data on variables like age group and gender were calculated as percentages and compared using the chi-square test. A p-value of <0.05 was considered to be statistically significant.
Out of 387 CSF samples, 32 (8.27%) were positive by culture for a bacterial pathogen. The proportion of meningococci among clinically suspected meningitis cases was 2.3% (9/387) among clinically suspected meningitis cases (Table 1). All N. meningitidis isolates belonged to serogroup A.
Table 1. Bacteria isolated from CSF culture (n=32).
|Type of Bacteria||Number||Percentage|
The clinically suspected meningitis cases were between 2 days and 89 years of age, with a median age of 12 months. The highest number of culture-positive bacterial meningitis cases was from the neonates. The meningococcal meningitis cases were between 5 days to 89 years old with a median age of 30 months. All except one meningococcal meningitis cases were below 15 years of age (Table 2).
|Age Group||Clinically Suspected Meningitis Cases
|Culture Positive Meningitis Cases
|Meningococcal Meningitis Cases
|Neonate < 1 month||128 (33.1)||13 (10.2)||1 (0.8)|
|Infant 1 month-1 year||74 (19.1)||4 (5.4)||1 (1.3)|
|Child 1-10 years||40 (10.3)||6 (15.0)||3 (7.5)|
|Adolescent 10-19 years||33 (8.5)||6 (18.2)||3 (9.1)|
|Adults 19-45 years||80 (20.7)||1 (1.2)||0 (0.00)|
|Adults above 45 years||32 (8.3)||2 (6.2)||1 (3.1)|
There was a slight predominance of clinically suspected male (56.8%) meningitis cases with male:female ratio of 1.32:1. Of the 9 meningococcal meningitis cases, 77.8% (7/9) were male (Table 3).
|Gender||Clinically Suspected Meningitis Cases
|Culture Positive Meningitis Cases
|Meningococcal Peningitis Cases
|p-value (Calculated Using Chi-square Test)|
|Female||167 (43.1)||14 (43.7)||2 (22.2)||0.93|
|Male||220 (56.8)||18 (56.2)||7 (77.8)|
The meningococcal isolates were susceptible to the commonly used antibiotics. All the meningococcal isolates were susceptible to ceftriaxone, chloramphenicol, meropenem and minocycline; however, 22% isolates showed resistance to cotrimoxazole and 11% intermediate resistant to ciprofloxacin (Table 4).
|Antibiotics||Susceptible %||Intermediate %||Resistant %|
The laboratory diagnosis of bacterial meningitis imposes a challenge. The actual estimate of bacterial meningitis in a resource-limited country like Nepal is difficult to establish. The proportion of culture positivity among 387 clinically suspected meningitis cases in this study (8.3%) was similar to other studies conducted in Nepal [7, 10]. The prevalence of bacterial meningitis varies worldwide, with reports from Asia, Europe and Africa [13-17]. A relatively lower prevalence (3-4.5%) of bacterial meningitis was reported by some researchers of our region [17-20]. The true burden of bacterial meningitis might have been underestimated due to the sole use of culture-based diagnostic methods. Though culture is considered as the gold standard for confirmation of cases, the positive rate is relatively low because of the fastidious and delicate nature of the major etiological agents and suboptimal storage and transportation conditions of specimens . Bacteria couldn't be isolated from 355 CSF samples in this study. This low culture yield of CSF might be partly due to the treatment of cases with antibiotics prior to the collection of samples, as 58% of cases were referred from other health care settings to our study sites, which are tertiary care centres. The incorporation of newer diagnostic methods such as latex agglutination and polymerase chain reaction are likely to improve the laboratory confirmation of clinical diagnosis. A hospital-based study in Kathmandu, Nepal, similar to our study reported 14% of confirmed bacterial meningitis cases by using molecular methods . Therefore, it is important to introduce molecular methods for surveillance of vaccine-preventable diseases, whenever possible.
With the introduction of and widespread use of vaccines, the etiology of bacterial meningitis has dramatically changed in the past few decades . Pneumococci, meningococci and H. influenzae were the most common bacterial pathogens isolated in this study. These findings are similar to the other reports on bacterial meningitis from Kathmandu, Nepal [18, 20, 22, 24, 25]. E.coli were isolated from neonates in our study. Few other studies in Nepal have documented the isolation of other gram-positive (Staphylococcus aureus, coagulase-negative S.aureus, alpha and beta haemolytic streptococci, viridians streptococci and enterococci) and gram-negative bacteria (Pseudomonas aeruginosa, Klebsiella pneumoniae, Aeromonas, Acinetobacter) from meningitis cases [7, 10, 20, 22]. Isolation of only four genera could be due to the exclusion of nosocomial, post-surgical and traumatic meningitis in this study.
Meningococcal meningitis epidemics have regularly recurred across the sub-Saharan African countries for over a century [26, 27]. To date, three studies explained the meningococcal epidemic (1982-1984) that occurred in the Kathmandu valley of Nepal. The first recorded meningitis epidemic caused by N. meningitidis serogroup A took place in April-May,1982 in Kathmandu valley of Nepal, resulting in 875 cases and 95 deaths . The epidemic continued until Feb 1984 and then declined after mass vaccination [5, 28]. In 1989, an intercontinental spread of meningitis epidemic occurred in Nepal along with Saudi Arabia and Chad and it was thought to be introduced through pilgrims on their return from the hajj . Since then, some hospital-based studies and case reports have reported meningococcal meningitis in Nepal [7, 10, 18-20, 22, 30, 31]. Reports of the low prevalence of meningococcal meningitis in India are comparable to our findings indicating geographical similarity [32, 33]. However, none of the hospital-based studies in Nepal have reported the information on circulating serogroups. Our study confirms that the circulating serogroup of N. meningitidis in Kathmandu, Nepal is serogroup A which has not changed over the past 35 years. Since meningococcal vaccination has not yet been included in the national immunization program and considering the epidemic potential of this organism, continuous surveillance should be done.
Similar to other studies, all except one meningococcal meningitis cases in this study were below 15 years [10, 18]. In contrast to the findings of previous studies, our study showed that bacterial meningitis in infants accounted for 53% of all cases [20, 34]. The incidence of meningitis in infants might be due to the vulnerability of their choroid plexus to penetration by bacteria during the septicemic process and to low immunological status . One meningococcal meningitis case in this study was 89 years of age. There is an increased multifactorial risk of bacterial meningitis for older adults, which includes both a greater susceptibility for underlying acute and chronic diseases and immunosenescence, a decline in immune function related to aging .
This study has demonstrated that meningitis is more common in males than in females. The male preponderance of 1.32: 1 in this study agrees with the findings in a tertiary hospital of Kathmandu . This male predominance might be due to the sex-related physiology and gender-specific behavior. There is a relative absence of gene locus for the elaboration of immunoglobulin, which is located on the X chromosomes .
The emergence and rapid spread of antibiotic-resistant isolates are of great concern globally. Fortunately, all the meningococcal isolates in this study were susceptible to ceftriaxone, chloramphenicol, meropenem and minocycline. This is in agreement with other studies conducted in Nepal [7, 18]. This offers the clinicians of our region a range of choice for empiric treatment in emergency situations. In contrast, there are previous reports of rare chloramphenicol-resistant N. meningitidis isolates in Nepal as well as in Australia, France, and Vietnam [10, 38, 39]. Similar to our study, cotrimoxazole resistance was reported in a tertiary care hospital of Nepal . One meningococcal isolate in our study was intermediate resistant to ciprofloxacin. Diminished fluoroquinolone (e.g., ciprofloxacin) susceptibility has been described only in few instances due to mutations in the gene (gyrA) encoding the gyrase A fluoroquinolone target [40, 41].
To the best of our knowledge, this is the first multicentric study on bacterial meningitis including the five major hospitals of our region. The strength of our study is that all age groups have been included at multicentric sites making the study population representative. The major limitation of our study is that laboratory diagnosis is based only on conventional culture-based methods, which might have contributed to the underestimation of actual disease burden.
The circulating N. meningitidis isolates in Kathmandu, Nepal is serogroup A which has not changed over the past 35 years. All isolates are susceptible to the commonly used antibiotics. The prevalence of meningococcal meningitis in Kathmandu, Nepal is low, but might have been underestimated due to the sole use of culture-based diagnostic methods. Therefore, detection of meningococci in CSF samples by alternative sensitive methods like PCR may be useful in the precise estimation of the actual disease burden.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
The Ethical approval of this study was obtained from the Nepal Health Research Council (Reg. No. 465/2016), Nepal. Parents or guardians were assured about the non-disclosure of information collected from them and were also informed about the use of data for analysis and using the results for improving patient care activities as well as publication without disclosing the name or identity of cases.
HUMAN AND ANIMAL RIGHTS
CONSENT FOR PUBLICATION
At the time of enrollment, consent was taken from the caregivers or guardians on behalf of the patients.
AVAILABILITY OF DATA AND MATERIALS
The authors confirm that the data supporting the findings of this research are available within the article.
We acknowledge the University Grants Commission, Nepal, for providing Ph.D. fellowship and PhD research support (PhD/73-74/S&T-01). The funding source does not have any role in study design, in the collection, analysis and interpretation of data; in the writing of the manuscript and in the decision to submit the manuscript for publication.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.
We are deeply thankful to all the patients participating in this study. We acknowledge all the concerned persons of Central Department of Microbiology, Tribhuvan University, Bhaktapur Hospital, Bir hospital, Kanti Children's Hospital, National Public Health Laboratory, Sukraraj Tropical and Infectious Diseases Hospital and Tribhuvan University Teaching Hospital for their contribution in sample and data collection in their respective units.
|||Jafri RZ, Ali A, Messonnier NE, et al. Global epidemiology of invasive meningococcal disease. Popul Health Metr 2013; 11(1): 17.
|||Crum-Cianflone N, Sullivan E. Meningococcal vaccinations. Infect Dis Ther 2016; 5(2): 89-112.
|||CDC. Meningococcal Disease. Centers for Disease Control and Prevention; 2016; Available from: . https://www.cdc.gov /meningococcal/index.html|
|||Vyse A, Wolter JM, Chen J, Ng T, Soriano-Gabarro M. Meningococcal disease in Asia: an under-recognized public health burden. Epidemiol Infect 2011; 139(7): 967-85. [Review].
|||Cochi SL, Markowitz LE, Joshi DD, et al. Control of epidemic group A meningococcal meningitis in Nepal. Int J Epidemiol 1987; 16(1): 91-7.
|||Sharma PR, Adhikari RK, Joshi MP, et al. Intravenous chloramphenicol plus penicillin versus intramuscular ceftriaxone for the treatment of pyogenic meningitis in Nepalese children. Trop Doct 1996; 26(2): 84-5.
|||Tiwari KB, Rijal B, Ghimire P, Sharma AP. Acute bacterial meningitis in Nepal. Nepal Med Coll J 2007; 9(2): 100-3.
|||Aggarwal M, Manchanda V, Talukdar B. Meningitis due to Neisseria meningitidis serogroup B in India. Indian Pediatr 2013; 50(6): 601-3.
|||Suri M, Kabra M, Singh S, Rattan A, Verma IC. Group B meningococcal meningitis in India. Scand J Infect Dis 1994; 26(6): 771-3.
|||Shrestha RG, Tandukar S, Ansari S, et al. Bacterial meningitis in children under 15 years of age in Nepal. BMC Pediatr 2015; 15: 94.
|||Maiden MC, Bygraves JA, Feil E, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 1998; 95(6): 3140-5.
|||Wayne P, Ed. CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement CLSI documen tM100-S24 2014.|
|||Jayaraman Y, Veeraraghavan B, Chethrapilly Purushothaman GK, et al. Hospital Based Sentinel Surveillance of Bacterial Meningitis (HBSSBM) Network Team. Burden of bacterial meningitis in India: Preliminary data from a hospital based sentinel surveillance network. PLoS One 2018; 13(5)e0197198
|||Jiang H, Su M, Kui L, et al. Prevalence and antibiotic resistance profiles of cerebrospinal fluid pathogens in children with acute bacterial meningitis in Yunnan province, China, 2012-2015. PLoS One 2017; 12(6)e0180161
|||Polkowska A, Toropainen M, Ollgren J, Lyytikäinen O, Nuorti JP. Bacterial meningitis in Finland, 1995-2014: A population-based observational study. BMJ Open 2017; 7(5)e015080
|||Ceyhan M, Gürler N, Ozsurekci Y, et al. Meningitis caused by Neisseria Meningitidis, Hemophilus Influenzae Type B and Streptococcus Pneumoniae during 2005-2012 in Turkey. A multicenter prospective surveillance study. Hum Vaccin Immunother 2014; 10(9): 2706-12.
|||Kambiré D, Soeters HM, Ouédraogo-Traoré R, et al. MenAfriNet Consortium. Nationwide trends in bacterial meningitis before the introduction of 13-Valent pneumococcal conjugate vaccine-burkina faso, 2011-2013. PLoS One 2016; 11(11): e0166384.
|||Ansari I, Pokhrel Y. Culture proven bacterial meningitis in children: Agents, clinical profile and outcome. Kathmandu Univ Med J (KUMJ) 2011; 9(33): 36-40. [KUMJ].
|||Kelly DF, Thorson S, Maskey M, et al. The burden of vaccine-preventable invasive bacterial infections and pneumonia in children admitted to hospital in urban Nepal. Int J Infect Dis 2011; 15(1): e17-23.
|||Pandey P, Jha B, Shrestha A. Etiology of meningitis from patients suspected of meningitis attending tribhuvan university teaching hospital, kathmandu, nepal. Current Research in Microbiology 2015; 6(2): 21-30.
|||Gray LD, Fedorko DP. Laboratory diagnosis of bacterial meningitis. Clin Microbiol Rev 1992; 5(2): 130-45.
|||Giri A, Arjyal A, Koirala S, et al. Aetiologies of central nervous system infections in adults in Kathmandu, Nepal: A prospective hospital-based study. Sci Rep 2013; 3: 2382.
|||VanDemark M. Acute bacterial meningitis: Current review and treatment update. Crit Care Nurs Clin North Am 2013; 25(3): 351-61.
|||Williams EJ, Thorson S, Maskey M, et al. Hospital-based surveillance of invasive pneumococcal disease among young children in urban Nepal. Clin Infect Dis 2009; 48(Suppl. 2): S114-22.
|||Shah AS, Knoll MD, Sharma PR, et al. Invasive pneumococcal disease in kanti children’s hospital, nepal, as observed by the South Asian Pneumococcal Alliance network. Clin Infect Dis 2009; 48(Suppl. 2): S123-8.
|||Mohammed I, Iliyasu G, Habib AG. Emergence and control of epidemic meningococcal meningitis in sub-Saharan Africa. Pathog Glob Health 2017; 111(1): 1-6.
|||Caugant DA, Kristiansen PA, Wang X, et al. Molecular characterization of invasive meningococcal isolates from countries in the African meningitis belt before introduction of a serogroup A conjugate vaccine. PLoS One 2012; 7(9)e46019
|||CDC. Epidemic meningococcal disease: recommendations for travelers to Nepal. MMWR Morb Mortal Wkly Rep 1985; 08;34(9): 119-20. 25|
|||Moore PS, Reeves MW, Schwartz B, Gellin BG, Broome CV. Intercontinental spread of an epidemic group A Neisseria meningitidis strain. Lancet 1989; 2(8657): 260-3.
|||Tuladhar R. Comparative evaluation of microscopic and cultural examination in bacterial meningitis among the patients attending Kanti Children Hospital [M.Sc dissertation]. 2007.|
|||Shrestha P, Shrestha NK, Giri S. Rapid recovery following fulminant meningococcemia complicated by myocarditis in a 15-year-old Nepalese girl: A case report. Int Med Case Rep J 2013; 6: 33-6.
|||Mani R, Pradhan S, Nagarathna S, Wasiulla R, Chandramuki A. Bacteriological profile of community acquired acute bacterial meningitis: A ten-year retrospective study in a tertiary neurocare centre in South India. Indian J Med Microbiol 2007; 25(2): 108-14.
|||Chinchankar N, Mane M, Bhave S, et al. Diagnosis and outcome of acute bacterial meningitis in early childhood. Indian Pediatr 2002; 39(10): 914-21.
|||Farag HF, Abdel-Fattah MM, Youssri AM. Epidemiological, clinical and prognostic profile of acute bacterial meningitis among children in Alexandria, Egypt. Indian J Med Microbiol 2005; 23(2): 95-101.
|||Townsend SM, Pollack HA, Gonzalez-Gomez I, Shimada H, Badger JL. Citrobacter koseri brain abscess in the neonatal rat: Survival and replication within human and rat macrophages. Infect Immun 2003; 71(10): 5871-80.
|||Choi C. Bacterial meningitis in aging adults. Clin Infect Dis 2001; 33(8): 1380-5.
|||Guerra-Silveira F, Abad-Franch F. Sex bias in infectious disease epidemiology: Patterns and processes. PLoS One 2013; 8(4)e62390
|||Shultz TR, Tapsall JW, White PA, et al. Chloramphenicol-resistant Neisseria meningitidis containing catP isolated in Australia. J Antimicrob Chemother 2003; 52(5): 856-9.
|||Galimand M, Gerbaud G, Guibourdenche M, Riou JY, Courvalin P. High-level chloramphenicol resistance in Neisseria meningitidis. N Engl J Med 1998; 339(13): 868-74.
|||Rosenstein NE, Perkins BA, Stephens DS, Popovic T, Hughes JM. Meningococcal disease. N Engl J Med 2001; 344(18): 1378-88.
|||Gorla MC, Pinhata JMW, Dias UJ, de Moraes C, Lemos AP. Surveillance of antimicrobial resistance in Neisseria meningitidis strains isolated from invasive cases in Brazil from 2009 to 2016. J Med Microbiol 2018; 67(6): 750-6.