Unearthing the Unknown Causes of Meningitis

Scientists around the world are painstakingly developing a road map to defeat meningitis by 2030. Once called “phrensy” or “brain fever,” meningitis can be caused by a multitude of pathogens. The three main culprits, and the primary focus of WHO surveillance platforms, are Haemophilus influenzae type b (Hib), Streptococcus pneumoniae, and Neisseria meningitidis. Before the advent of antibiotics, meningitis by these pathogens killed almost all children they affected; this changed drastically with timely use of appropriate antibiotics starting in the mid-1900s. Next, with the global rollout of the Hib vaccine beginning in the 1990s, rates of Hib meningitis have significantly reduced in most countries. Pneumococcal and meningococcal vaccines are also being introduced inmanypopulations, andobvious positive impacts are emerging. However, as these vaccines are taken up by populations and meningitis surveillance systems are strengthened with incorporation of new technologies, we are uncovering large gaps in the current meningitis literature, specifically related to incomplete microbiological diagnosis of suspected meningitis cases. In an article published in this issue of the Journal, Kwambana-Adams and team suggest roles for several additional bacterial andviral pathogens in causingmeningitis in< 5-yearold children. Using 711 cerebrospinal fluid (CSF) specimens collected from five different countries across West Africa, Senegal, Ghana, Togo, Nigeria and Niger, this study showed that a large proportion of meningitis cases remained microbiologically undiagnosed after molecular testing (triplex PCR) for the three pathogens noted previously. To extend surveillance for many additional potential pathogens, the team used a customized TaqMan Array Card (TAC) to simultaneously detect 15 bacteria, 17 viruses, one fungus, and two protozoans using quantitative polymerase chain reaction (qPCR). With this approach, microbiological diagnosis of probable meningitis causes was increased by 18% compared with triplex PCR (from 2% to 20%). Among the 19 different pathogens detected, Escherichia coli, Plasmodium spp, S. pneumoniae, Staphylococcus aureus, and Klebsiella pneumoniae were the most common, followed by several viruses including parvovirus B19 and dengue. All detected pathogens have previously been associated with neurological infections, but are not commonly screened using molecular techniques, highlighting the importance of this study. The epidemiological profiles of some of these pathogens were different than anticipated; for example, Gram-negative pathogens like E. coli and K. pneumoniae are usually associated with neonatal meningitis, but the authors detected them in older children. In addition, in about 4% of the specimens, more than one pathogen was detected in the CSF, a phenomenon not commonly reported in the literature. A proportion of these “uncommon” findings may be explained by underlying conditions like severe malnutrition found in the study populations. It is also noteworthy that most of the previous epidemiological data on meningitis come from developed nations, where demographic and clinical profiles of cases are markedly different. Although very important and timely, the findings of this study should be considered within the context of several limitations. Thiswasa retrospectiveetiologydetectionstudy, not specifically designed to collect and preserve specimens for sensitive molecular testing by the TAC technology. Lack of stringent collectionandpreservationmethodsmayhavehampered thequalityof the specimens, asDNAandRNAare susceptible todegradation, and as contaminationmay have been introduced. The 711CSF samples were gathered from five countries with different demographic profiles of cases, making comparison between countries and generalization of the results difficult, if not impossible. Finally, and perhaps most importantly, there were no control (healthy/truenegative) specimens included in thestudy.A large study that used TACs to test more than 10,000 blood and respiratory samples to determine causes of neonatal sepsis depicted the importance of including healthy control specimens and rigorous statistical modeling to distinguish between true pathogens and false positives. This caveat is also evident in this study; DNA of pathogenic bacteria and parasites were detected and considered an etiology in many cases with very low white blood cell count (< 10/cmm), whereas specimens from cases of bacterial meningitis usually have higher white blood cell count. With techniques as sensitive as the qPCR, the risk of false positives is high and should be considered with utmost diligence to avoid misinterpretations. A recent study from Bangladesh that sequenced total nucleic acid present in about 100 CSF samples of childrenwith andwithoutmeningitis illustrated the abundance of “pathogenic” bacterial nucleic acid on the skin of the children, in hospital and laboratory environments, and in testing reagents, further illustrating the importance of incorporating control specimens to negate false positives. These limitations should be critically considered when designing future studies. Despite these limitations, this study demonstrates key gaps in our knowledge on etiology of meningitis and proposes a molecular technique that can be incorporated into ongoing and futuresurveillancestudies.With increasinguseofover-the-counter antibiotics, culture-positivity rates are declining, and, hence, molecular techniques todetect bacterial etiologies arebecoming increasingly important. Furthermore,most surveillance studies in resource-poor settings do not screen for viruses. A technique that canprobe formanydifferent pathogens simultaneouslymay play a vital role in comprehensive attribution of etiologies of meningitis. However, the panel of pathogens to be screened for in such a test will likely differ between countries/populations, as *Address correspondence to Senjuti Saha, Child Health Research Foundation, 5th Floor, SEL Huq Skypark, 23/2 Khilji Road, Block-B, Mohammadpur, Dhaka 1207, Bangladesh. E-mail: senjutisaha@ chrfbd.org